Rice grain with reduced ROS1A activity

ABSTRACT

The present invention relates to rice grain with thickened aleurone. Also provided is a rice plant comprising at least one genetic variation which reduces the activity of at least one ROS1a gene in the plant. Grain of the invention, or aleurone therefrom, has improved nutritional properties, and hence is particularly useful for human and animal feed products.

FIELD OF THE INVENTION

The present invention relates to rice grain with thickened aleurone.Also provided is a rice plant comprising at least one genetic variationwhich reduces the activity of at least one ROS1a gene in the plant.Grain of the invention, or aleurone therefrom, has improved nutritionalproperties, and hence is particularly useful for human and animal feedproducts.

BACKGROUND OF THE INVENTION

Worldwide, cereal grains such as wheat, rice, maize and to a lesserextent barley, oats and rye are the major source of human caloric intakefrom the starch content of the grain. Cereal grain is also important insupplying other nutritional components such as protein, vitamins,minerals and dietary fibre. Different parts of the grains contributedifferently for these nutritional components. Starch is stored in thestarchy endosperm of cereal grains, whereas the other nutritionalcomponents are more concentrated in the embryo and bran (Buri et al.,2004). However, the bran is often removed before use in food,particularly in rice which is then eaten as white rice.

Cereal grain develops from double fertilisation events between maternaland paternal gametophytes. One of two sperm cells from the pollen tubefuses with an egg to produce a zygote that develops into an embryo, andthe other sperm cell fuses with the diploid central cell of themegagametophyte to produce a primary endosperm nucleus, from which thegenetically triploid endosperm develops. Thus, the endosperm includingthe aleurone is triploid, having two copies of the maternal haploidgenome and one copy of the paternal haploid genome. In dicotyledonousseeds, the endosperm is consumed by the developing embryo whereas inmonocotyledons such as rice the endosperm persists to make up the bulkof the mature grain.

The mature endosperm of cereals has four cell types with distinctcharacteristics, namely the starchy endosperm which is characterised byits abundant contents of starch granules and storage proteins, theepidermal-like aleurone which is most often one cell layer in thicknesssurrounding most of the starchy endosperm, transfer cells at the base ofthe seed over the main maternal vasculature, and a layer ofembryo-surrounding cells which form a lining for the embryo early ingrain development but later may only surround the suspensor whichconnects the embryo and starchy endosperm (Becraft et al., 2001a). Theembryo forms within a cavity within the starchy endosperm. Cerealaleurone tissue therefore comprises the outermost layer(s) of theendosperm in cereal grains, and surrounds the starchy endosperm and partof the embryo.

Aleurone cells are distinguished from starchy endosperm cells by theirmorphology, biochemical composition and gene expression profiles(Becraft and Yi, 2011). Aleurone cells are generally oil andprotein-rich and secrete enzymes allowing the mobilization of endospermreserves during seed germination. Each aleurone cell is enclosed withina fibrous cell wall that is thicker than endosperm cell walls and thatis composed mainly of arabinoxylans and beta glucans in various ratiosand are highly autofluorescent. The aleurone layer is the only layer ofthe endosperm that in cereals is sometimes pigmented with anthocyanins.

Cereal aleurone is only one cell layer in thickness in wheat andwild-type maize (Buttrose 1963; Walbot, 1994), mostly one but up tothree cell layers in the dorsal region of the endosperm in rice(Hoshikawa, 1993), and three cell layers in wild-type barley (Jones,1969). In normal endosperm, the aleurone is extremely regular and thepatterns of cell division are highly organised. Wild-type maturealeurone cells are nearly cuboid in section with a dense cytoplasmincluding granules, small vacuoles and inclusion bodies made of protein,lipid and phytin or of protein plus carbohydrate. In mature cerealgrains, the aleurone is the only endosperm tissue that remains alive,although in a dormant, desiccated form. Upon imbibition, the embryoproduces gibberellins which induce synthesis of amylases and otherhydrolases by the aleurone which are released into the starchy endospermto break down storage compounds to form sugars and amino acids for earlygrowth of the embryo into a seedling.

The regulation of aleurone development in cereal grains has beenreviewed by Becraft and Yi (2011). Multiple levels of genetic regulationcontrol aleurone cell fate, differentiation and organisation, and manygenes are involved in the processes, only some of which have beenidentified. For example, maize defective kernal1 (dek1) loss-of-functionmutants have no aleurone layer indicating that the wild-type Dek1polypeptide is required for specifying the outer cell layer as aleurone(Becraft et al., 2002). The Dek1 polypeptide is a large integralmembrane protein with 21 membrane-spanning domains and a cytoplasmicdomain containing an active calpain protease. Another gene in maize,CRINKLY4 (CR4) encodes a receptor kinase that functions as a positiveregulator of aleurone fate, and cr4 mutants have reduced aleurone(Becraft et al., 2001 b).

Several instances of thickened aleurones in cereal grain mutants havebeen reported in the literature, but none have proven useful because ofpleiotrophic effects, or agronomic and production problems.

Shen et al. (2003) reported the identification of maize mutants in thesupernumary aleurone layers1 (sal1) gene which in different mutants had2-3 or up to seven layers of aleurone cells instead of the normal singlelayer. The SAL1 polypeptide was identified as a class E vacuolar sortingprotein. Homozygous sal1-1 mutant grain had defective embryos thatfailed to germinate and had much reduced starchy endosperm. A lesscomplete mutant that was homozygous for the sal1-2 allele exhibited a 2cell-layer aleurone. However, the mutant plants grew to a height of only30% of the wild-type, had a reduced root mass and were poor in seedsetting (Shen et al. 2003). These plants were not agronomically useful.

Yi et al. (2011) reported the identification of a thick aleurone1 (thk1)mutant in maize. The mutant kernals showed a multilayer aleurone.However, the mutant kernals lacked well-developed embryos and did notgerminate when sown. The wild-type Thk1 gene encoded a Thk1 polypeptidewhich acted downstream of the Dek1 polypeptide which was required foraleurone development in maize (Becraft et al., 2002).

A maize extra cell layer (Xcl) gene mutant was identified by its effecton leaf morphology. It produced a double aleurone layer as well asmultilayered leaf epidermis (Kessler et al., 2002). The Xcl mutation wasa semi-dominant mutation that disrupted cell division anddifferentiation patterns in maize, producing thick and narrow leaveswith an abnormal shiny appearance.

Maize mutants in the disorgal1 and disorgal2 (dil1 and dil2) genesexhibited aleurones having a variable number of layers with cells ofirregular shapes and sizes (Lid et al., 2004). However, homozygous dil1and dil2 mutant grains were shrunken due to reduced accumulation ofstarch, and the mature mutant grains germinated at low rates and did notdevelop into viable plants.

In barley, elo2 mutants showed similarly disorganised cells andirregularities of the aleurone layers, resulting from aberrantpericlinal cell division (Lewis et al., 2009). The plants also showedincreased cell layers in the leaf epidermis, with bulging and distortedcells on the epidermis. Importantly, the homozygous mutant plants weredwarfed, producing grain weight of less than 60% of wild-type, and werenot useful for grain production.

In rice, two transcription factors that control the expression of seedstorage proteins also influence aleurone cell fate (Kawakatsu et al.,2009). Reduction in expression by co-suppression constructs of a geneencoding a rice prolamin box binding factor (RPBF) polypeptide, which isin the DOF zinc finger transcription factor class, resulted in asporadic multilayered aleurone consisting of large, disordered cells.There was also a significant reduction in seed storage proteinexpression and accumulation, and starch and lipids were accumulated atsubstantially reduced levels. Expression of the rice homologs of themaize Dek1, CR4 and SAL1 genes was also reduced, showing that the RPBFand RISBZ1 factors operated in the same regulatory pathway as thosegenes.

Demethylation of DNA

In a completely different area of plant science, demethylation of DNA isnow summarised. Plants methylate some cytosine nucleotides in nuclearDNA at carbon 5 of the pyrimidine ring, forming 5-methylcytosine(5-meC). The methylated cytosine may occur in any of three contexts,namely CG, CHG (where H=A, C or T) and CHH methylation, each catalysedby a different methyltransferase. At least in Arabidopsis thaliana andprobably in most plants including rice (Zemach et al., 2010), CGmethylation is catalysed by enzymes in the Methyltransferase 1 (Met1)family, CHG methylation is catalysed by methylases in theChromomethylase family, and CHH methylation occurs through anRNA-mediated reaction catalysed by Domains Rearranged Methylases (DRM)using small RNAs as guide sequences (Law and Jacobsen, 2010). Cytosinemethylation, which occurs in only a small proportion of all cytosines,most often occurs in heterochromatic DNA and in regions rich inrepetitive DNA and transposons, suppressing their activity. It alsooccurs in transcribed regions of the nuclear DNA, including in promoterregions of genes, and is thereby involved in the control of expressionof many genes.

Cytosine methylation of DNA is reversible through demethylation, whichmay happen passively through DNA replication or actively through theactivity of demethylation enzymes. One pathway for active demethylationof DNA in plants is through a base excision repair (BER) pathway whichuses DNA glycosylase enzymes. These enzymes remove 5-meC from thedouble-stranded DNA backbone and then cleave the DNA backbone (lyaseactivity) at the abasic site by successive β- and δ-eliminationreactions. The repair is completed by insertion of an unmethylatedcytosine nucleotide by a DNA polymerase activity.

There are four 5-meC DNA glycosylase/lyases in the Arabidopsis genome,designated Demeter (DME), Demeter-like 2 (DML2), Demeter-like 3 (DML3)and Repressor of Silencing (ROS1). Genetic and biochemical analysesshowed that all four function as DNA demethylases (Gong et al., 2002;Agius et al., 2006; Morales-Ruiz et al., 2006), with DME functioningprimarily in the egg cell and endosperm and the others functioning inother tissues. Other plants similarly show a multiplicity ofdemethylases (Zemach et al., 2010).

There is a need for rice grain having thickened aleurone from plants,particularly rice plants that are also phenotypically normal andagronomically useful.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides grain of a rice plant, thegrain comprising an aleurone, a starchy endosperm, a ROS1a gene encodinga ROS1a polypeptide and (i) one or more genetic variations which eachreduce the activity of at least one ROS1a gene in the plant whencompared to a corresponding wild-type rice plant, and/or (ii) saidaleurone is thickened compared to aleurone from a correspondingwild-type grain.

In an embodiment, the ROS1a polypeptide has DNA glycosylase activity. Inan embodiment, the ROS1a polypeptide has a level of DNA glycosylaseactivity which is between 2% and about 60% of the level of DNAglycosylase activity of a corresponding wild-type ROS1a polypeptideand/or of ROS1a polypeptide whose amino acids have a sequence set forthin SEQ ID NO: 2.

In another embodiment, the ROS1a polypeptide is a variant of acorresponding wild-type ROS1a polypeptide in that their amino acidsequences are different. In a preferred embodiment, the rice graincomprises a ROS1a (Ta2) variant polypeptide which has an amino acidsequence provided as SEQ ID NO:1, the polypeptide being a variant of thewild-type ROS1a polypeptide whose amino acid sequence is provided as SEQID NO:2.

In an embodiment, the grain has a level of ROS1a polypeptide between 2%and about 60% of that present in the grain compared to the level ofROS1a polypeptide in the corresponding wild-type grain.

In an embodiment, the thickened aleurone comprises at least two, atleast three, at least four or at least five layers of cells, about 3,about 4, about 5 or about 6 layers of cells, or 2-8, 2-7, 2-6 or 2-5layers of cells. In an embodiment, the grain is from a rice plant andthe thickened aleurone comprises 5-8, 5-7, 5-6 or 2-5 layers of cells.In an embodiment, the aleurone layer is increased in thickness comparedto the aleurone of a wild-type rice grain by about 100%, or about 150%or about 200%, or about 250%.

The genetic variation, preferably an introduced genetic variation, whichreduces the activity of at least one ROS1a gene in the plant can be anytype of genetic manipulation which reduces or impairs the production ofwild-type levels of ROS1a polypeptide in the rice grain. Examples ofsuch genetic variations include, but are not necessarily limited to,

(a) a ROS1a gene encoding a mutant ROS1a polypeptide with reduced DNAglycosylase activity relative to the wild-type ROS1a polypeptide (SEQ IDNO:2);

(b) a ROS1a gene which when expressed produces a reduced level of awild-type ROS1a polypeptide, for example which comprises a splice-sitemutation that results in a reduced level of expression of the ROS1agene, relative to the wild-type ROS1a gene whose cDNA sequence isprovided as SEQ ID NO:8, or which ROS1a gene comprises a mutation in itspromoter which results in reduced expression of the ROS1a gene relativeto the wild-type ROS1a gene;

(c) an exogenous nucleic acid construct which encodes a polynucleotidewhich reduces expression of a ROS1a gene in the rice plant, preferablywherein the nucleic acid construct comprises a DNA region encoding thepolynucleotide operably linked to a promoter which is expressed indeveloping grain of the rice plant at least at a time point between thetime of anthesis and 7 days post-anthesis, and

(d) a ROS1a gene comprising a premature translational stop codon in itsprotein coding region such that the gene encodes, but may or may notproduce, a truncated polypeptide relative to a wild-type ROS1 apolypeptide.

In a preferred embodiment, the rice grain comprises (i) an aleurone,(ii) a starchy endosperm, (iii) a ROS1a gene which comprises a geneticvariation which reduces the activity of the ROS1a gene in a rice plantcompared to a wild-type ROS1a gene, wherein said ROS1a gene whichcomprises the genetic variation encodes a variant ROS1a polypeptiderelative to SEQ ID NO:2, and wherein the aleurone is thickened comparedto aleurone from a corresponding wild-type rice grain. In an embodiment,the genetic variation in the ROS1a gene is an introduced geneticmodification. In a preferred embodiment, the variant ROS1a polypeptideis different in amino acid sequence to the sequence provided as SEQ IDNO:2 at least by an insertion or deletion of one or more amino acids oran amino acid substitution relative to SEQ ID NO:2. In an even morepreferred embodiment, the variant ROS1a polypeptide is different inamino acid sequence to the sequence provided as SEQ ID NO:2 by aninsertion of one or more amino acids or by a single amino acidsubstitution relative to SEQ ID NO:2, such as, for example, one of theamino acid substitutions as listed in Table 3. The rice grain may havebeen treated so that it is no longer able to germinate such as, forexample, having been cooked, or it may not have been so treated suchthat it is able to germinate and grow and thereby provide a rice plantof the invention. In a most preferred embodiment, the aleurone of therice grain is pigmented such as, for example, the rice grain is blackrice as defined herein.

In another preferred embodiment, the rice grain comprises (i) analeurone, (ii) a starchy endosperm, (iii) a ROS1a gene which comprises agenetic variation which reduces the activity of the ROS1a gene in a riceplant compared to a wild-type ROS1a gene, wherein said ROS1a gene whichcomprises the genetic variation encodes a ROS1a polypeptide whose aminoacid sequence is the same as a wild-type ROS1a polypeptide such as, forexample, SEQ ID NO:2, wherein said ROS1a gene is expressed in a riceplant at a reduced level relative to a wild-type ROS1a gene, wherein thealeurone is thickened compared to aleurone from a correspondingwild-type rice grain. In an embodiment, the genetic variation in theROS1a gene is an introduced genetic modification which results in theROS1a gene being expressed at the reduced level. In an embodiment, thegenetic variation is selected from the group consisting of (i) asplice-site mutation that results in a reduced level of expression ofthe ROS1a gene, relative to the wild-type ROS1a gene whose cDNA sequenceis provided as SEQ ID NO:8, (ii) a ROS1a gene promoter mutation whichresults in reduced expression of the ROS1a gene relative to thewild-type ROS1a gene, and (iii) an exogenous nucleic acid molecule,preferably integrated into the nuclear genome of the rice plant, whichencodes an RNA polynucleotide which reduces expression of a ROS1a genein the rice plant. The rice grain may have been treated so that it is nolonger able to germinate such as, for example, having been cooked, or itmay not have been so treated such that it is able to germinate and growand thereby give rise to a rice plant of the invention. In a mostpreferred embodiment, the aleurone of the rice grain is pigmented suchas, for example, the rice grain is black rice as defined herein.

In an embodiment, the rice plant has a level of DNA glycosylase activityin its developing grain which is between 2% and about 60% of the levelof DNA glycosylase activity in a corresponding wild-type developinggrain. In a preferred embodiment, the rice plant has a level of DNAglycosylase activity in its developing grain which is between 2% and50%, or between 2% and 40%, or between 2% and 30%, or between 2% and 20%of the level of DNA glycosylase activity in a corresponding wild-typedeveloping grain.

In an embodiment, the activity of at least one ROS1a gene in the riceplant is reduced in one or more or all of aleurone, pericarp, nucellarprojection, ovary, testa and starchy endosperm of the developing grain.

In an embodiment, the activity of a ROS1a gene is reduced at least at atime point between the time of anthesis and 7 days post-anthesis and/orin the egg cell prior to anthesis. In an embodiment, the exogenousnucleic acid molecule may be operably linked to a promoter which isexpressed at least at a time point between the time of anthesis and 7days post-anthesis, such that the encoded RNA polynucleotide reducesexpression of the ROS1a gene in the rice plant during that time.

In an embodiment, the rice plant is male and female fertile.

In an embodiment, the rice plant exhibits delayed grain maturation. Inan embodiment, the grain maturation is delayed by 2-10 days or 2-15 daysrelative to a wild-type rice plant.

In an embodiment, the grain comprises, when compared to a correspondingwild-type grain, one or more or all of the following, each on a weightbasis,

-   -   i) a higher mineral content, preferably the mineral content is        the content of one or more or all of zinc, iron potassium,        magnesium, phosphorus and sulphur,    -   ii) a higher antioxidant content,    -   iii) a higher phytate content,    -   iv) a higher content of one or more or all of vitamins B3, B6        and B9,    -   v) a higher dietary fibre content and/or insoluble fibre        content,    -   vi) a starch content which is between about 90% and about 100%        by weight relative to the starch content of the corresponding        wild-type grain;    -   vii) a higher sucrose content,    -   viii) a higher monosaccharide content, and    -   ix) a lipid content of at least 90% or at least 100% relative to        the lipid content of the corresponding wild-type grain. In an        embodiment, the content of the component is increased by 10-50%        or preferably 10-100% relative to the corresponding content in a        wild-type rice grain.

In an embodiment, the grain comprises an embryo.

In an embodiment, the grain is whole grain or cracked grain.

In an embodiment, the grain has been processed so that it is no longerable to germinate, preferably by heat treatment. For example, the grainhas been cooked with water at 100° C. for at least 5 minutes. In anembodiment, the grain is cracked grain or milled grain.

In an alternative embodiment, the grain has a germination rate which isbetween about 70% and about 100% relative to the germination rate of acorresponding wild-type grain. That is, a collection of at least 100grains has an average germination rate of 70-100% relative to wild-type.When the grains germinate and grow, rice plants of the invention areproduced.

In an embodiment, the grain comprises a ROS1a gene which encodes a ROS1apolypeptide which has DNA glycosylase activity, preferably in one ormore of aleurone, testa and starchy endosperm of the grain, wherein theROS1a polypeptide which has DNA glycosylase activity is preferably amutant ROS1a polypeptide.

In another embodiment, the grain comprises a mutant ROS1a polypeptidehaving decreased DNA glycosylase activity when expressed in the riceplant compared to a corresponding wild-type ROS1a polypeptide,preferably wherein the mutant ROS1a polypeptide comprises one or moreamino acid substitutions, deletions or insertions which reduces DNAglycosylase activity compared to the corresponding wild-type ROS1apolypeptide. The mutant ROS1a polypeptide may have no DNA glycosylaseactivity, provided the grain comprises another ROS1a polypeptide whichhas DNA glycosylase activity. For example, the ROS1a gene may encode twoROS1a polypeptides through alternative splicing, one of which has DNAglycosylase activity whereas the other does not. In a preferredembodiment, the mutant ROS1a polypeptide has an insertion of seven aminoacids relative to the wild-type ROS1a polypeptide, such as for examplethe mutant ROS1a polypeptide whose amino acid sequence is provided asSEQ ID NO:1.

In a further embodiment, the grain has a reduced total amount of ROS1apolypeptide compared to a corresponding wild-type grain, preferablyreduced in one or more of aleurone, testa and starchy endosperm of thegrain, provided that the grain comprises a ROS1a gene which encodes aROS1a polypeptide which has DNA glycosylase activity. The total amountof ROS1a polypeptide may also be decreased in the pollen of the riceplant.

In another embodiment, the genetic variation, preferably introducedgenetic variation, is an exogenous nucleic acid construct which encodesa polynucleotide which reduces expression of a ROS1a gene in the riceplant, preferably reduced in the rice plant at least at a time pointbetween the time of anthesis and 7 days post-anthesis, provided that thegrain comprises at least one ROS1a gene which encodes a ROS1apolypeptide which has DNA glycosylase activity.

In an embodiment, the grain is pigmented in its outer layer(s), forexample the grain is brown grain or black grain of the rice plant, thegrain comprising (i) an aleurone having a thickness of at least 2 celllayers, or 2-7 cell layers, and (ii) a mutant ROS1a gene which encodes aROS1a polypeptide which comprises one or more amino acid substitutions,insertions or deletions which reduces DNA glycosylase activity whencompared to a corresponding wild-type rice ROS1a polypeptide, thereduced DNA glycosylase activity occurring in the rice plant at least ata time point between the time of anthesis and 7 days post-anthesis,provided that at least at a time point between the time of anthesis and7 days post-anthesis the rice plant has between 2% and about 60% of thelevel of DNA glycosylase activity in developing grain compared to thewild-type rice plant.

In an embodiment, the grain is pigmented in its outer layer(s), forexample the grain is brown grain or black grain of a rice plant, thegrain comprising (i) an aleurone having a thickness of at least 2 celllayers, or 2-7 cell layers, and (ii) an exogenous nucleic acid constructwhich encodes a polynucleotide which reduces expression of a ROS1a genein the rice plant, wherein the exogenous nucleic acid constructcomprises a DNA region encoding the polynucleotide operably linked to apromoter which is expressed in developing grain of the rice plant atleast at a time point between the time of anthesis and 7 dayspost-anthesis, such that at least at a time point between the time ofanthesis and 7 days post-anthesis the rice plant has between 2% andabout 60% of the level of DNA glycosylase activity in the developinggrain compared to the wild-type rice plant. In a preferred embodiment,the promoter is a promoter other than a constitutive promoter, such as,for example, a promoter which is expressed in the endosperm ofdeveloping seed. In a most preferred embodiment, the promoter is an LTPpromoter.

In another embodiment, the ROS1a polypeptide comprises amino acids whosesequence is at least 95% identical to SEQ ID NO: 2, or the ROS1apolypeptide(s) comprises amino acids whose sequence is at least 95%identical, at least 97.5% identical, or at least 99% identical, to SEQID NO: 2 and which sequence is different to the amino acid sequence ofthe corresponding wild-type ROS1a polypeptide.

In an embodiment, the ROS1a polypeptide comprises one or more or all ofthe following motifs; DHGSIDLEWLR (SEQ ID NO: 44), GLGLKSVECVRLLTLHH(SEQ ID NO: 45), AFPVDTNVGRI (SEQ ID NO: 46), VRLGWVPLQPLPESLQLHLLE (SEQID NO: 47), ELHYQMITFGKVFCTKSKPNCN (SEQ ID NO: 48) and HFASAFASARLALP(SEQ ID NO: 49).

The present invention also provides a population of rice grains, each ofwhich comprises the same genetic variation(s), the same ROS1a gene, thesame ROS1a polypeptide and/or has the same characteristics as describedin the above embodiments. That is, the population is genetically and/orphenotypically uniform. The population of such rice grains may beobtained or derived from a single progenitor rice plant or grain, forexample may be derived at least 2, at least 3 or at least 4 progenygenerations from a progenitor plant or grain.

The present inventors have identified variant ROS1a polypeptides withreduced DNA glycosylase activity. Thus, in another aspect the presentinvention provides a purified and/or recombinant ROS1a polypeptide whoseamino acid sequence is different to the amino acid sequence of acorresponding wild-type ROS1a polypeptide and which has reduced,preferably no, DNA glycosylase activity when compared to thecorresponding wild-type ROS1a polypeptide.

In an embodiment, the purified and/or recombinant ROS1a polypeptidecomprises amino acids having a sequence which is at least 95% identical,at least 97.5% identical, or at least 99% identical, to SEQ ID NO: 2.

In another aspect, the present invention provides an isolated and/orexogenous polynucleotide encoding a ROS1a polypeptide of the invention.

In a further aspect, the present invention provides an isolated and/orexogenous polynucleotide which, when present in a rice plant, reducesthe expression of a ROS1a gene.

The skilled person is well aware of different types of polynucleotidesthat can be used to reduce the expression of a target gene, and howthese polynucleotides can be designed. Examples include, but are notlimited to, an antisense polynucleotide, a sense polynucleotide, acatalytic polynucleotide, a microRNA, a double stranded RNA (dsRNA)molecule or a processed RNA product thereof.

In an embodiment, the polynucleotide is a dsRNA molecule, or a processedRNA product thereof, comprising at least 19 consecutive nucleotideswhich is at least 95% identical to the complement of SEQ ID NO: 7 or 8(where thymine (T) is uracil (U)), or at least 95% identical to thecomplement an mRNA encoding a ROS1a polypeptide whose amino acidsequence is provided as SEQ ID NO: 1 or 2.

In another embodiment, the dsRNA molecule is a microRNA (miRNA)precursor and/or wherein the processed RNA product thereof is a miRNA.

In an embodiment, the polynucleotide is used for reducing the expressionof a ROS1a gene in developing grain of a rice plant at least at a timepoint between the time of anthesis and 7 days post-anthesis.

In a further aspect, the present invention provides nucleic acidconstruct and/or vector encoding a polynucleotide of the invention,wherein the nucleic acid construct or vector comprises a DNA regionencoding the polynucleotide operably linked to a promoter which isexpressed in developing grain of a rice plant at least at a time pointbetween the time of anthesis and 7 days post-anthesis.

In a further aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide of the invention, or a nucleicacid construct and/or vector of the invention.

In an embodiment, the cell is a rice plant cell such as a cell of ricegrain, preferably a rice aleurone.

In an embodiment, the exogenous polynucleotide, nucleic acid constructor vector is integrated into the genome of the cell, preferably into thenuclear genome.

Also provided is a cell of a rice plant comprising a ROS1a gene encodinga ROS1a polypeptide and a genetic variation, preferably an introducedgenetic variation, which reduces the activity of at least one ROS1a genein the cell when compared to a corresponding wild-type cell.

In an embodiment, the cell is an aleurone, pericarp, nucellarprojection, ovary, testa or starchy endosperm cell.

In another aspect, the present invention provides a rice plant, or apopulation of rice plants, which produces grain of the invention, apolypeptide of the invention, a polynucleotide of the invention, anucleic acid construct and/or vector of the invention and/or whichcomprises a cell of the invention. In an embodiment, each of the riceplants

Also provided is a population of at least 100, or at least 1,000, riceplants of the invention growing in a field. In a preferred embodiment,the rice plants in the field are mostly (>50%), preferably all, riceplants of the invention. In a most preferred embodiment, the at least100, or at least 1,000, rice plants are genetically and/orphenotypically the same, for example comprising the same geneticvariation.

In another aspect, the present invention provides a method of producinga cell of the invention, the method comprising a step of introducing anexogenous polynucleotide of the invention, or a nucleic acid constructand/or vector of the invention, into a cell, preferably a rice cell.

In a further aspect, the present invention provides a method ofproducing a rice plant of the invention or transgenic grain therefrom,the method comprising the steps of

i) introducing into a rice cell, an exogenous polynucleotide of theinvention, or a nucleic acid construct and/or vector of the invention,

ii) obtaining a transgenic rice plant from a cell obtained from step i),the transgenic rice plant being transgenic for the exogenouspolynucleotide, nucleic acid construct or vector or part thereof, and

iii) optionally harvesting grain from the plant of step ii), the grainbeing transgenic for the exogenous polynucleotide, nucleic acidconstruct or vector, and

iv) optionally producing one or more generations of transgenic progenyplants from the transgenic grain, the progeny plants being transgenicfor the exogenous polynucleotide, nucleic acid construct or vector,thereby producing the rice plant or transgenic grain.

In another aspect, the present invention provides a method of producinga rice plant of the invention or grain therefrom, the method comprisingthe steps of

i) introducing into a rice cell, a mutation of an endogenous ROS1a genesuch that the mutated ROS1a gene encodes a ROS1a polypeptide of theinvention, or does not encode a ROS1a polypeptide,

ii) obtaining a rice plant from a cell obtained from step i), the riceplant comprising the mutation of the endogenous ROS1a gene, and

iii) optionally harvesting grain from the plant of step ii), the graincomprising the mutation of the endogenous ROS1a gene, and

iv) optionally producing one or more generations of progeny plants fromthe grain, the progeny plants comprising the mutation of the endogenousROS1a gene, thereby producing the rice plant or grain.

In an embodiment, the rice plant or grain comprises at least one ROS1agene which encodes a ROS1a polypeptide which has DNA glycosylaseactivity.

In a further aspect, the present invention provides a method ofselecting a rice plant or rice grain of the invention, the methodcomprising the steps of

i) screening a population of rice plants or grain each of which wereobtained from a mutagenic treatment of progenitor rice cells, grain orplants, for the production of grain of the invention or for the presenceof a mutation in a ROS1a gene, or the presence of rice grain of theinvention, and

ii) selecting from the population of step (i) a rice plant whichproduces grain of the invention or which comprises a mutant ROS1a gene,or rice grain of step (i) which is rice grain of the invention, therebyselecting the rice plant or grain.

The method may comprise a step of producing one or more progeny plantsor grain from the selected rice plant, or at least two generations ofprogeny plants, and/or harvesting grain from progeny plants. Preferably,the progeny plants are homozygous for the genetic variation.

In a further aspect, the present invention provides a method ofselecting a rice plant of the invention, the method comprising the stepsof

i) producing one or more progeny plants from rice grain, the rice grainhaving been derived from a cross of two parental rice plants,

ii) screening the one or more progeny plants of step i) for theproduction of grain of the invention, and

iii) selecting a progeny plant which produces the grain,

thereby selecting the rice plant. In a preferred embodiment, the ricegrain is black rice grain.

In an embodiment, screening step i) or step ii) comprises one or more orall of:

i) analysing a sample comprising DNA from a progeny plant for thegenetic variation,

ii) analysing the thickness of aleurone of grain obtained from a progenyplant, and

iii) analysing the nutritional content of the grain or a part thereof.

Preferably, the genetic variation is an introduced genetic variation.

In an embodiment, step iii) comprises one or more or all of:

i) selecting a progeny plant which is homozygous for the geneticvariation, wherein the genetic variation reduces DNA glycosylaseactivity in the rice plant when compared to a corresponding wild-typerice plant,

ii) selecting a progeny plant whose grain has an increased aleuronethickness compared to a corresponding wild-type grain,

iii) selecting a progeny plant whose grain or a part thereof has analtered nutritional content compared to a corresponding wild-type grainor part thereof.

In a further embodiment, the method further comprises

i) crossing two parental rice plants, preferably wherein one of theparental rice plants produces grain of the invention, or

ii) backcrossing one or more progeny plants from step i) with plants ofthe same genotype as a first parental rice plant which does not producegrain of the invention for a sufficient number of times to produce aplant with a majority of the genotype of the first parental rice plantbut which produces grain of the invention, and

iii) selecting a progeny plant which produces grain of the invention.

Also provided is a rice plant and rice grain, and products therefrom,produced using a method of the invention.

Further provided is the use of an exogenous polynucleotide of theinvention, or a nucleic acid construct and/or vector of the invention,to produce a recombinant cell, a transgenic rice plant or transgenicgrain.

In an embodiment, the use is to produce rice grain of the invention.

In a further aspect, the present invention provides a method foridentifying a rice plant which produces grain of the invention, themethod comprising the steps of

i) obtaining a nucleic acid sample from a rice plant, and

ii) screening the sample for the presence or absence of a geneticvariation which reduces the activity of a ROS1a gene in the plant whencompared to a corresponding wild-type rice plant.

In an embodiment, the genetic variation is one or both of

a) a nucleic acid construct expressing a polynucleotide, or thepolynucleotide encoded thereby, which when present in a rice plantreduces the expression of a ROS1a gene, and

b) a gene, or mRNA encoded thereby, which expresses a mutant ROS1apolypeptide with reduced ROS1a polypeptide activity.

In an embodiment, the presence of the genetic variation indicates thatgrain of the plant has a thickened aleurone when compared to acorresponding plant lacking the genetic variation(s).

In yet another aspect, the present invention provides a method foridentifying a rice plant which produces grain of the invention, themethod comprising the steps of

i) obtaining grain from a rice plant, and

ii) screening the grain or a portion thereof for one or more of

-   -   a) a thickened aleurone,    -   b) the amount of ROS1a polypeptide and/or activity in the grain,        and    -   c) the amount of mRNA encoded by ROS1a genes in the grain.

In an embodiment, the method identifies a rice plant of the invention.

In another aspect, the present invention provides a method of producinga rice plant part, preferably grain, the method comprising,

a) growing a rice plant, or at least 100 such rice plants in a field, ofthe invention, and

b) harvesting the rice plant part from the rice plant or rice plants.

In a further aspect, the present invention provides a method ofproducing rice flour, bran, wholemeal, malt, starch or oil obtained fromgrain, the method comprising;

a) obtaining grain of the invention, and

b) processing the grain to produce the flour, bran, wholemeal, malt,starch or oil.

In another aspect, the present invention provides a product producedfrom grain of the invention, or a rice plant of the invention, or from apart of said grain or rice plant.

In an embodiment, the product comprises one or more or all of the ROS1agene, the genetic variation (preferably introduced genetic variation),the exogenous nucleic acid construct and the thickened aleurone.

In an embodiment, the part is rice bran.

In an embodiment, the product is a food ingredient, beverage ingredient,food product or beverage product. Examples include, but are not limitedto,

i) the food ingredient or beverage ingredient is selected from the groupconsisting of wholemeal, flour, bran, starch, malt and oil,

ii) the food product is selected from the group consisting of: leavenedor unleavened breads, pasta, noodles, animal fodder, breakfast cereals,snack foods, cakes, pastries and foods containing a flour-based sauce,or

iii) the beverage product is a packaged beverage or a beveragecomprising ethanol.

In a further aspect, the present invention provides a method ofpreparing a food or beverage ingredient of the invention, the methodcomprising processing grain of the invention, or bran, flour, wholemeal,malt, starch or oil from the grain, to produce the food or beverageingredient.

In another aspect, the present invention provides a method of preparinga food or beverage product of the invention, the method comprisingmixing grain of the invention, or bran, flour, wholemeal, malt, starchor oil from the grain, with another food or beverage ingredient.Preferably, the weight of the grain, bran, flour, wholemeal, malt,starch or oil that is used in the method is at least 10% on a weightbasis relative to the food product.

Also provided is the use of grain of the invention or part thereof, or arice plant of the invention or part thereof, as animal feed or food, orto produce feed for animal consumption or food for human consumption.

In a further aspect, the present invention provides a compositioncomprising one or more of a polypeptide of the invention, apolynucleotide of the invention, a nucleic acid construct and/or vectorof the invention, or a cell of the invention, and one or more acceptablecarriers.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1. Schematic diagram of the map-based cloning of the ta2 gene.Numbers below each line show the number of recombinants in the mappingpopulation which displayed recombination between the marker and the ta2gene. The solid bars on the third line show the extent of the codingregions on the rice chromosome. The solid bars on the fourth line showthe protein coding regions in the gene; the intervening lines representthe introns (intron 1 is in the 5′UTR and not shown here). The asteriskshows the position of the ta2 mutation in intron 14 at position Chr1:6451738 with reference to the rice genome sequence.

FIG. 2. Nucleotide sequences of cDNAs corresponding to mRNAs obtainedfrom developing grains of wild-type (WT) and ta2 genotypes. Dashes inthe WT and two of the ta2 sequences indicate absence of nucleotides.Most of the ta2 mRNAs had 21 nucleotide insertions. The wild-typesequence is SEQ ID NO: 10, the mutant sequence is SEQ ID NO: 11.

FIG. 3. Predicted amino acid sequences from cDNAs corresponding to mRNAsobtained from developing grains of wild-type (upper amino acid sequence,SEQ ID NO: 12) and ta2 (lower sequence, SEQ ID NO: 13). Dashes in the WTsequence indicate absence of amino acids opposite the seven amino acidinsertion (CSNVMRQ; SEQ ID NO: 14) in the ta2 polypeptide. Stars belowthe sequences indicate that the same amino acids were present in thewild-type and mutant polypeptides at those positions.

FIG. 4. Amino acid sequence alignment of Arabidopsis DME (NM001085058.1)(SEQ ID NO:6), Arabidopsis ROS1a (NM129207.4) (SEQ ID NO:50) and riceROS1a homologs (SEQ ID NO:2). Asterisks below the alignment representamino acid positions which are conserved in all three polypeptides.Semi-colons represent fully conservative amino acid changes, whereassingle dots represent partially conservative amino acid changes.

FIG. 5. Amino acid sequence alignment Arabidopsis ROS1a (NM129207.4)(SEQ ID NO:50) and rice ROS1a homologs (SEQ ID NO:2). Asterisks belowthe alignment represent amino acid positions which are conserved in allthree polypeptides. Semi-colons represent fully conservative amino acidchanges, whereas single dots represent partially conservative amino acidchanges.

FIG. 6. Real-time RT-PCR results showing relative expression of the riceTA2 gene in multiple tissues.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1—Rice ROS1a mutant (Ta2) polypeptide.

SEQ ID NO: 2—Wild-type rice ROS1a polypeptide.

SEQ ID NO: 3—Wild-type rice ROS1b polypeptide.

SEQ ID NO: 4—Wild-type rice ROS1c polypeptide.

SEQ ID NO: 5—Wild-type rice ROS1d polypeptide.

SEQ ID NO: 6—Arabidopsis DEMETER polypeptide.

SEQ ID NO: 7—Full length cDNA encoding rice ROS1a mutant (Ta2)polypeptide. Open reading frame spans nucleotides 341 to 6220.

SEQ ID NO: 8—Full length cDNA encoding rice ROS1a polypeptide. Openreading frame spans nucleotides 341 to 6199.

SEQ ID NO: 9—Rice ROS1a gene. Promoter and 5′UTR: nucleotides 1-4726,translation start codon ATG 4727-4729; translation stop codon TAG15867-15869; 3′-UTR from 15870-16484, downstream of the gene from16485-16885. Nucleotide positions of introns are: intron 1, 7494-7724;2, 7816-7909; 3, 9426-9571; 4, 9652-10452; 5, 10538-10628; 6,10721-10795; 7, 10865-10951; 8, 10989-11069; 9, 11153-11834; 10,12282-12385; 11, 12423-12508; 12, 12567-12650; 13, 12791-13017; 14,13084-13201; 15, 13317-14668; 16, 14708-15732-11006. The sequenceincludes 401 nucleotides at the 3′ end which does not form part of thegene.

SEQ ID NO: 10—Partial wild type rice ROS1a cDNA sequence provided inFIG. 2.

SEQ ID NO: 11—Partial mutant (Ta2) rice ROS1a cDNA sequence provided inFIG. 2.

SEQ ID NO: 12—Partial wild type rice ROS1a protein sequence provided inFIG. 3.

SEQ ID NO: 13—Partial mutant (Ta2) rice ROS1a protein sequence providedin FIG. 3.

SEQ ID NO: 14—Additional amino acids in ROS1a mutant (Ta2) polypeptidewhen compared to wild type (SEQ ID NO:2).

SEQ ID NOs 15 to 43—Oligonucleotide primers.

SEQ ID NO's 44 to 49—Highly conserved amino acid motifs within theglycosylase domain of the wild-type rice ROS1a polypeptide.

SEQ ID NO: 50—Arabidopsis ROS1a polypeptide.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, plant molecular biology, protein chemistry, andbiochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term “about”, unless stated to the contrary, refersto +/−10%, more preferably +/−5%, more preferably +/−2.5%, even morepreferably +/−1%, of the designated value. The term “about” includes theexact designated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

Selected Definitions

The terms “aleurone” and “aleurone layer” are used interchangeablyherein. The aleurone layer is the outermost layer of the endosperm ofrice grain, distinct from the inner starchy endosperm, and surrounds thestarchy endosperm and part of the embryo. The cells that make up thealeurone layer are therefore the outermost cells of the endosperm, thestarchy component of the grain. While it is technically part of theendosperm, sometimes referred to as the peripheral endosperm, thealeurone is considered part of the bran from a practical standpoint asit is removed with the pericarp and testa layers of the bran. Unlikecells of the starchy endosperm, aleurone cells remain alive at grainmaturity. The aleurone layer is an important part of the nutritionalvalue of rice grain comprising minerals, vitamins such as vitamin A andB group vitamins, phytochemicals, and fiber.

Embodiments of the invention relate to a range of number of “layers ofcells”, at least in part because at any one cross sectional point ofgrain of the invention, the layers of cells observed at any single pointwithin the cross section, or between cross sections, may vary to someextent. More specifically, an aleurone with, for example, seven layersof cells may not have the seven layers surrounding the entire innerstarchy endosperm but has seven layers surrounding at least half of theinner starchy endosperm.

The term “thickened” when used in relation to aleurone of grain of theinvention is a relative term used when comparing grain of the inventionto a corresponding wild-type grain. Aleurone of grain of the inventionhas an increased number of cells and/or increased number of layers ofcells when compared to aleurone of corresponding wild-type grain. Thealeurone is thereby increased in thickness as measured in m. In anembodiment, the thickness is increased by at least 50%, preferably by atleast 100%, and may be increased by as much as 500% or 600%, eachpercentage being relative to the thickness of the aleurone of acorresponding wild-type grain, and understanding each percentage to bethe average increase over the ventral side of the grain and preferablyover the whole grain. In an embodiment, the thickness of the aleuronelayer is determined across an entire cross section of the grain. In anembodiment, the thickness of the aleurone is determined by at leastanalysis on the ventral side of the grain. In another embodiment,thickened aleurone of grain of the invention comprises cells of varyingsize and irregular orientation compared to that of correspondingwild-type grain where the aleurone generally has regularly orientedrectangular cells.

Polypeptides

As used herein, the term “ROS1 polypeptide” refers to a member of aprotein family of DNA glycosylase related molecules which are related inamino acid sequence to SEQ ID NOs: 1 to 5 in that they are at least 95%identity to one or more of the amino acid sequences set forth in SEQ IDNOs: 1 to 5. ROS1 polypeptides include the ROS1a, ROS1b, ROS1c and ROS1dpolypeptides of wild-type rice, including naturally occurring variantsand mutant forms thereof. ROS1 polypeptides include such polypeptidesfound in wild-type rice plants as well as variants thereof producedeither artificially or found in nature, such as found in either Indicaand Japonica rice plants, and either have or do not have DNA glycosylaseactivity, including ROS1 polypeptides which have some DNA glycosylaseactivity but at a reduced level compared to a corresponding wild-typeROS1 polypeptide. Examples of ROS1 polypeptides of wild-type rice plantsinclude those which have the amino acid sequence set forth in one of SEQID NOs: 2 to 5, as well as variant polypeptides which have an amino acidsequence which is at least 95%, at least 97%, or preferably at least 99%identical to one or more of the amino acid sequences set forth in SEQ IDNOs: 2 to 5 and which are found in nature. As used herein, ROS1polypeptides do not include Demeter (DME) polypeptides which are arelated DNA glycosylase family which are much less than 95% identical inamino acid sequence to SEQ ID NOs:1 to 5.

As used herein, the term “ROS1a polypeptide” means a DNA glycosylaserelated molecule whose amino acid sequence is at least 95% identical toSEQ ID NO: 2, preferably at least 97% or more preferably at least 99%identical to SEQ ID NO:2. ROS1a polypeptides include the polypeptidesfound in wild-type rice plants as well as variants thereof producedeither artificially or found in nature, and either have or do not haveDNA glycosylase activity, provided they have the required level of aminoacid sequence identity to SEQ ID NO:2. For example, the ROS1apolypeptide whose sequence is provided as SEQ ID NO:1 is thought to haveno DNA glycosylase activity, yet it is a ROS1a polypeptide as definedherein. In a preferred embodiment, the ROS1a polypeptide has some DNAglycosylase activity but at a reduced level compared to the wild-typeROS1a polypeptide whose amino acid sequence is provided as SEQ ID NO:2.For example, Table 3 lists ROS1a mutant polypeptides which are thoughtto have reduced DNA glycosylase activity.

The skilled person can readily use known techniques to distinguish aROS1a polypeptide from other structurally related proteins such as otherROS1 polypeptides, specifically from ROS1b, ROS1c and ROS1 dpolypeptides, for example, using in silico phylogenetic analysis orprotein alignments. A ROS1 a polypeptide can therefore be identified asa ROS1a polypeptide based on structural features alone. For example, seeFIGS. 4 and 5 herein. A ROS1a polypeptide of the invention may or maynot have DNA glycosylase activity, or may have reduced DNA glycosylaseactivity when compared to a wild-type ROS1a polypeptide such as one withthe amino acid sequence set forth in SEQ ID NO: 2.

As used herein, the term “which sequence is different to the amino acidsequence of the corresponding wild-type ROS1a polypeptide”, or similarphrases, are comparative terms where the amino acid sequence of a ROS1apolypeptide of the invention is different to the amino acid sequence ofthe protein from which it is derived and/or most closely related thatexists in nature. In an embodiment, the amino acid sequence of the ROS1apolypeptide has one or more insertions, deletions or amino acidsubstitutions (or a combination of these) relative to the correspondingwild-type amino acid sequence. The ROS1a polypeptide may have 2, 3, 4, 5or 6-10 amino acid substitutions relative to the corresponding wild-typeROS1a polypeptide. In a preferred embodiment, the ROS1a polypeptide hasonly a single insertion, single deletion or single amino acidsubstitution relative to the corresponding wild-type polypeptide. Inthis context, the “single insertion” and “single deletion” includeswhere multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), contiguousamino acids are inserted or deleted, respectively and the “correspondingwild-type polypeptide” means the wild-type polypeptide from which thevariant is derived and/or the natural polypeptide to which the variantis most closely related. The ROS1a polypeptide may be a truncated ROS1apolypeptide which may be encoded by, for example, a ROS1a gene whichcomprises a premature translational stop codon in the protein openreading frame relative to the wild-type ROS1a gene from which it isderived, or the ROS1a polypeptide may be full-length i.e. having thesame number of amino acid residues as the corresponding wild-type ROS1apolypeptide. An example of a naturally occurring (wild-type) ROS1apolypeptide is one whose amino acid sequence is set forth as SEQ ID NO:2, and examples of variant ROS1a polypeptides which have only a singleinsertion, deletion or amino acid substitution are given in SEQ ID NO:1and Table 3.

As used herein, the term “DNA glycosylase activity” refers to an enzymeinvolved in base excision repair (classified under EC number EC 3.2.2).The enzyme typically also has DNA lyase activity, in which the DNA baseis excised and the backbone DNA strand is cleaved. In an embodiment,“DNA glycosylase activity” as used in the context of the presentinvention relates to active demethylation where 5-methylcytosineresidues are excised and replaced with unmethylated cytosines. In apreferred embodiment, a ROS1a polypeptide of the invention with DNAglycosylase activity has at least five identifiable motifs. One is ahelix-hairpin-helix (HhH) motif (for example, amino acids 1491-1515 inSEQ ID NO:2 or a homologous amino acid sequence). Another is aglycine/proline-rich motif followed by a conserved aspartic acid (GPD),and four conserved cysteine residues (in the region of amino acids1582-1598 of SEQ ID NO:2) to hold a [4Fe-4S] cluster in place. There isalso a lysine-rich domain (for example, amino acids 87-139 in SEQ IDNO:2 or a homologous amino acid sequence). Unlike other members of theHhH DNA glycosylase superfamily members, ROS1a polypeptide-familymembers contain two additional conserved domains (domains A and B)flanking the central glycosylase domain. In the rice ROS1a polypeptide(SEQ ID NO:2), domain A occurs at amino acids 859 to 965, theglycosylase domain occurs at amino acids 1403 to 1616, and domain Boccurs at amino acids 1659 to 1933. Domain A contains a repetitivemixed-charge cluster at amino acids 882-892. DNA glycosylase activitycan be measured using standard techniques known in the art, such asdescribed in Example 8.

Highly conserved amino acid motifs within the glycosylase domain of thewild-type rice ROS1a polypeptide include DHGSIDLEWLR (SEQ ID NO: 44,amino acids 1467-1477 in SEQ ID NO:2), GLGLKSVECVRLLTLHH (SEQ ID NO: 45,amino acids 1493-1509 in SEQ ID NO:2), AFPVDTNVGRI (SEQ ID NO: 46, aminoacids 1511-1521 in SEQ ID NO:2), VRLGWVPLQPLPESLQLHLLE (SEQ ID NO: 47,amino acids 1523-1543 in SEQ ID NO:2), ELHYQMITFGKVFCTKSKPNCN (SEQ IDNO: 48, amino acids 1569-1590 in SEQ ID NO:2) and HFASAFASARLALP (SEQ IDNO: 49, amino acids 1600-1613 in SEQ ID NO:2). One or two amino acidsubstitutions may occur in these motifs, or not. Other conserved aminoacids can be readily identified by aligning the amino acid sequences forwild-type rice ROS1a (SEQ ID NO:2) with the DME polypeptide fromArabidopsis thaliana (SEQ ID NO:6) and/or the A. thaliana ROS1apolypeptide (see FIGS. 4 and 5). Further guidance regarding theidentification of conserved amino acids can be obtained from Kapazoglouet al. (2012).

As used herein, the terms “which reduces DNA glycosylase activitycompared to the corresponding wild-type ROS1a polypeptide”, “which hasreduced, preferably no, DNA glycosylase activity when compared to thecorresponding wild-type ROS1a polypeptide”, or similar phrases, arerelative terms where the DNA glycosylase activity of a variant/mutantROS1a polypeptide is lower than the protein from which it is derivedand/or most closely related that exists in nature. For instance, as theskilled person would appreciate, the rice Ta2 mutant described herein(SEQ ID NO:1) has reduced DNA glycosylase activity when compared to thecorresponding wild-type rice ROS1a polypeptide (SEQ ID NO:2). Otherexamples of ROS1a polypeptides with reduced DNA glycosylase activitycomprise mutations/variations corresponding to the amino acids describedin Table 3 which confer a thickened aleurone phenotype such assubstituting the serine at an amino acid position corresponding to aminoacid number 156 of SEQ ID NO:2 with another amino acid such as aphenylalanine, substituting the serine at an amino acid positioncorresponding to amino acid number 214 of SEQ ID NO:2 with another aminoacid such as a phenylalanine, substituting the serine at an amino acidposition corresponding to amino acid number 1413 of SEQ ID NO:2 withanother amino acid such as an asparagine, substituting the alanine at anamino acid position corresponding to amino acid number 441 of SEQ IDNO:2 with another amino acid such as a valine, substituting the serineat an amino acid position corresponding to amino acid number 1357 of SEQID NO:2 with another amino acid such as a phenylalanine, substitutingthe lysine at an amino acid position corresponding to amino acid number501 of SEQ ID NO:2 with another amino acid such as a serine, andsubstituting the arginine at an amino acid position corresponding toamino acid number 482 of SEQ ID NO:2 with another amino acid such as alysine. In a particularly preferred embodiment, the grain has at leastsome DNA glycosylase activity, preferably some ROS1a DNA glycosylaseactivity, because evidence suggests that the absence of DNA glycosylaseactivity in the egg cell and early in seed development is lethal to riceplants. In an embodiment, the grain has between about 30% and 98%, orbetween about 40% and 98%, or between about 40% and 90%, or betweenabout 40% and 85%, or between about 40% and 80%, less DNA glycosylaseactivity when compared to grain from a corresponding isogenic plantlacking an genetic variation (preferably introduced genetic variation)which reduces DNA glycosylase activity in the grain.

By “substantially purified polypeptide” or “purified polypeptide” wemean a polypeptide that has generally been separated from the lipids,nucleic acids, other peptides, and other contaminating molecules withwhich it is associated in its native state. Preferably, thesubstantially purified polypeptide is at least 90% free from othercomponents with which it is naturally associated. In an embodiment, thepolypeptide of the invention has an amino acid sequence which isdifferent to a naturally occurring ROS1a polypeptide i.e. is an aminoacid sequence variant, as defined above.

Grain, plants and host cells of the invention may comprise an exogenouspolynucleotide encoding a polypeptide of the invention. In theseinstances, the grain, plants and cells produce a recombinantpolypeptide. The term “recombinant” in the context of a polypeptiderefers to the polypeptide encoded by an exogenous polynucleotide whenproduced by a cell, which polynucleotide has been introduced into thecell or a progenitor cell by recombinant DNA or RNA techniques such as,for example, transformation. Typically, the cell comprises anon-endogenous gene that causes an altered amount of the polypeptide tobe produced. In an embodiment, a “recombinant polypeptide” is apolypeptide made by the expression of an exogenous (recombinant)polynucleotide in a plant cell.

The terms “polypeptide” and “protein” are generally usedinterchangeably.

The % identity of a polypeptide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 1,000 aminoacids in length, and the GAP analysis aligns the two sequences over aregion of at least 1,000 amino acids. More preferably, the querysequence is at least 1,250 amino acids in length and the GAP analysisaligns the two sequences over a region of at least 1,250 amino acids.More preferably, the query sequence is at least 1,500 amino acids inlength and the GAP analysis aligns the two sequences over a region of atleast 1,500 amino acids. Even more preferably, the GAP analysis alignstwo sequences over their entire length, which for a ROS1a polypeptide isabout 1,800 to 2,100 amino acid residues.

With regard to a defined polypeptide, it will be appreciated that %identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide comprises anamino acid sequence which is at least 95%, more preferably at least 96%,more preferably at least 97%, more preferably at least 98%, morepreferably at least 99%, more preferably at least 99.1%, more preferablyat least 99.2%, more preferably at least 99.3%, more preferably at least99.4%, more preferably at least 99.5%, more preferably at least 99.6%,more preferably at least 99.7%, more preferably at least 99.8%, and evenmore preferably at least 99.9% identical to the relevant nominated SEQID NO.

As used herein, the phrase “at a position corresponding to amino acidnumber” or variations thereof refers to the relative position of theamino acid compared to surrounding amino acids. In this regard, in someembodiments a polypeptide of the invention may have deletional orsubstitutional mutation which alters the relative positioning of theamino acid when aligned against, for instance, SEQ ID NO: 2. Determininga corresponding amino acid position between two closely related proteinsis well within the capability of the skilled person.

Amino acid sequence mutants of the polypeptides of the present inventioncan be prepared by introducing appropriate nucleotide changes into anucleic acid of the present invention, or by in vitro synthesis of thedesired polypeptide. Such mutants include, for example, deletions,insertions or substitutions of residues within the amino acid sequence.A combination of deletion, insertion and substitution can be made toarrive at the final construct, provided that the final peptide productpossesses the desired characteristics. Preferred amino acid sequencemutants have only one, two, three, four or less than 10 amino acidchanges relative to the reference wildtype polypeptide. Mutantpolypeptides of the invention have reduced “ROS1a polypeptide activity”when compared to a corresponding wild-type naturally occurring ROS1apolypeptide such as a polypeptide which comprises amino acids having asequence set forth as SEQ ID NO: 2.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rational designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey have reduced ROS1a polypeptide activity, such as reduced DNAglycosylase activity, when compared to one or more or all of a ROS1apolypeptide which comprises amino acids having a sequence provided asSEQ ID NO: 2. For instance, the method may comprise producing atransgenic plant expressing the mutated/altered DNA and determining i)the effect of the mutated/altered DNA on aleurone thickness and ii)whether a ROS1a gene has been mutated/altered.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries, e.g., by (1) substituting with non-conservative amino acidchoices, (2) deleting the target residue, or (3) inserting otherresidues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide molecule removed and a different residue inserted in itsplace. Where it is not desirable to maintain a certain activity, or toreduce a certain activity, it is preferable to make non-conservativesubstitutions, particularly at amino acid positions which are highlyconserved in the relevant protein family. Examples of conservativesubstitutions are shown in Table 1, and hence non-conservativesubstitutions will be those not shown in Table 1.

In an embodiment a mutant/variant polypeptide has one or two or three orfour amino acid changes when compared to a naturally occurringpolypeptide. In a preferred embodiment, the changes are in one or moreof the motifs which are highly conserved between the different ROS1apolypeptides provided herewith, particularly in known conservedstructural domains. As the skilled person would be aware, such changescan reasonably be predicted to alter the activity of the polypeptidewhen expressed in a cell.

The primary amino acid sequence of a polypeptide of the invention can beused to design variants/mutants thereof based on comparisons withclosely related enzymes, particularly DNA glycosylases. As the skilledaddressee will appreciate, residues highly conserved amongst closelyrelated proteins are more likely to be able to be altered, especiallywith non-conservative substitutions, and activity reduced than lessconserved residues (see above).

TABLE 1 Conservative substitutions. Original Conservative ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

Also included within the scope of the invention are polypeptides of thepresent invention which are differentially modified after synthesis,e.g., by post-translational modification in a cell, for example byphosphorylation, which may modulate its activity.

Polynucleotides and Genes

The present invention refers to various polynucleotides. As used herein,a “polynucleotide” or “nucleic acid” or “nucleic acid molecule” means apolymer of nucleotides, which may be DNA or RNA, and includes genomicDNA, mRNA, cRNA, dsRNA, and cDNA. It may be DNA or RNA of cellular,genomic or synthetic origin, for example made on an automatedsynthesizer, and may be combined with carbohydrate, lipids, protein orother materials, labelled with fluorescent or other groups, or attachedto a solid support to perform a particular activity defined herein, orcomprise one or more modified nucleotides not found in nature, wellknown to those skilled in the art. The polymer may be single-stranded,essentially double-stranded or partly double-stranded. Basepairing asused herein refers to standard basepairing between nucleotides,including G:U basepairs. “Complementary” means two polynucleotides arecapable of basepairing (hybridizing) along part of their lengths, oralong the full length of one or both. A “hybridized polynucleotide”means the polynucleotide is actually basepaired to its complement. Theterm “polynucleotide” is used interchangeably herein with the term“nucleic acid”. Preferred polynucleotides of the invention encode apolypeptide of the invention.

By “isolated polynucleotide” we mean a polynucleotide which hasgenerally been separated from the polynucleotide sequences with which itis associated or linked in its native state, if the polynucleotide isfound in nature. Preferably, the isolated polynucleotide is at least 90%free from other components with which it is naturally associated, if itis found in nature. Preferably the polynucleotide is not naturallyoccurring, for example by covalently joining two shorter polynucleotidesequences in a manner not found in nature (chimeric polynucleotide).

The present invention involves reduction of gene activity and theconstruction and use of chimeric genes. As used herein, the term “gene”includes any deoxyribonucleotide sequence which includes a proteincoding region or which is transcribed in a cell but not translated, aswell as associated non-coding and regulatory regions. Such associatedregions are typically located adjacent to the coding region or thetranscribed region on both the 5′ and 3′ ends for a distance of about 2kb on either side. In this regard, the gene may include control signalssuch as promoters, enhancers, termination and/or polyadenylation signalsthat are naturally associated with a given gene, or heterologous controlsignals in which case the gene is referred to as a “chimeric gene”. Thesequences which are located 5′ of the coding region and which arepresent on the mRNA are referred to as 5′ non-translated sequences. Thesequences which are located 3′ or downstream of the coding region andwhich are present on the mRNA are referred to as 3′ non-translatedsequences. The term “gene” encompasses both cDNA and genomic forms of agene.

An “allele” refers to one specific form of a genetic sequence (such as agene) within a cell, an individual plant or within a population, thespecific form differing from other forms of the same gene in thesequence of at least one, and frequently more than one, variant siteswithin the sequence of the gene. The sequences at these variant sitesthat differ between different alleles are termed “variances”, or“polymorphisms”. A “polymorphism” as used herein denotes a variation inthe nucleotide sequence between alleles at a genetic locus of theinvention, of different species, cultivars, strains or individuals of aplant. A “polymorphic position” is a preselected nucleotide positionwithin the sequence of the gene at which the sequence difference occurs.In some cases, genetic polymorphisms cause an amino acid sequencevariation within a polypeptide encoded by the gene, and thus apolymorphic position can result in the location of a polymorphism in theamino acid sequence at a predetermined position in the sequence of thepolypeptide. In other instances, the polymorphic region may be in anon-polypeptide encoding region of the gene, for example in the promoterregion and may thereby influence expression levels of the gene. Typicalpolymorphisms are deletions, insertions or substitutions. These caninvolve a single nucleotide (single nucleotide polymorphism or SNP) ortwo or more nucleotides.

As used herein, a “mutation” is a polymorphism which produces aphenotypic change in the plant or a part thereof. As known in the art,some polymorphisms are silent, for example a single nucleotide change ina protein coding region which does not change the amino acid sequence ofthe encoded polypeptide due to the redundancy of the genetic code. Adiploid plant will typically have one or two different alleles of asingle gene, but only one if both copies of the gene are identical i.e.the plant is homozygous for the allele. Polyploid plants generally havemore than one homoeolog of any particular gene. For instance, hexaploidwheat has three subgenomes (often referred to as “genomes”) designatedthe A, B and D genomes, and therefore has three homoeologs of most ofits genes, one in each of the A, B and D genomes.

The term “ROS1a gene encoding a ROS1a polypeptide” or “ROS1a gene” asused herein refers to a nucleotide sequence which encodes a ROS1apolypeptide as defined herein. The ROS1a gene may be an endogenousnaturally occurring gene, or comprise a genetic variation (preferably anintroduced genetic variation) as defined herein. A ROS1a gene encoding aROS1a polypeptide in grain of the invention may or may not have introns.In one example, the grain of the invention is from rice and at least oneallele of an ROS1a gene encodes a ROS1a polypeptide with reduced DNAglycosylase activity when compared to a ROS1a polypeptide from acorresponding a wild type rice plant (such as which comprises a sequenceof amino acids as provided in SEQ ID NO: 2). An example of such a ROS1apolypeptide with reduced DNA glycosylase activity is the rice Ta2 mutant(SEQ ID NO:1).

As used herein, the phrase “or inactivation of a ROS1a gene” or“reduction of expression of a ROS1a gene” or variations thereof refersto any genetic variation which reduces (partially), or completelyprevents, the expression of the gene encoding a functional ROS1apolypeptide. Such genetic variations include mutations in the promoterregion of the gene which reduce transcription of the gene beingtranscribed, for example by using gene editing to delete or substitutenucleotides from the promoter of the ROS1a gene, or intron splicingmutations which alter the amount or position of splicing to form mRNA.

A genomic form or clone of a gene containing the transcribed region maybe interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences”, which may be eitherhomologous or heterologous with respect to the “exons” of the gene. An“intron” as used herein is a segment of a gene which is transcribed aspart of a primary RNA transcript but is not present in the mature mRNAmolecule. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA). “Exons” as used herein refer to the DNA regions corresponding tothe RNA sequences which are present in the mature mRNA or the mature RNAmolecule in cases where the RNA molecule is not translated. An mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide. The term “gene” includes a synthetic orfusion molecule encoding all or part of the proteins of the inventiondescribed herein and a complementary nucleotide sequence to any one ofthe above. A gene may be introduced into an appropriate vector forextrachromosomal maintenance in a cell or, preferably, for integrationinto the host genome.

As used herein, a “chimeric gene” refers to any gene that comprisescovalently joined sequences that are not found joined in nature.Typically, a chimeric gene comprises regulatory and transcribed orprotein coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. In anembodiment, the protein coding region of an ROS1a gene is operablylinked to a promoter or polyadenylation/terminator region which isheterologous to the ROS1a gene, thereby forming a chimeric gene. In analternate embodiment, a gene encoding a polynucleotide which, whenpresent in grain of a rice plant, down regulates the production and/oractivity of a ROS1a polypeptide in the grain is operably linked to apromoter or polyadenylation/terminator region which is heterologous tothe polynucleotide, thereby forming a chimeric gene.

The term “endogenous” is used herein to refer to a substance that isnormally present or produced in an unmodified plant at the samedevelopmental stage as the plant under investigation. An “endogenousgene” refers to a native gene in its natural location in the genome ofan organism. As used herein, “recombinant nucleic acid molecule”,“recombinant polynucleotide” or variations thereof refer to a nucleicacid molecule which has been constructed or modified by recombinant DNAtechnology. The terms “foreign polynucleotide” or “exogenouspolynucleotide” or “heterologous polynucleotide” and the like refer toany nucleic acid which is introduced into the genome of a cell byexperimental manipulations.

Foreign or exogenous genes may be genes that are inserted into anon-native organism, native genes introduced into a new location withinthe native host, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure. The term“genetically modified” includes introducing genes into cells bytransformation or transduction, mutating genes in cells and altering ormodulating the regulation of a gene in a cell or organisms to whichthese acts have been done or their progeny.

Furthermore, the term “exogenous” in the context of a polynucleotide(nucleic acid) refers to the polynucleotide when present in a cell thatdoes not naturally comprise the polynucleotide. The cell may be a cellwhich comprises a non-endogenous polynucleotide resulting in an alteredamount of production of the encoded polypeptide, for example anexogenous polynucleotide which increases the expression of an endogenouspolypeptide, or a cell which in its native state does not produce thepolypeptide. Increased production of a polypeptide of the invention isalso referred to herein as “over-expression”. An exogenouspolynucleotide of the invention includes polynucleotides which have notbeen separated from other components of the transgenic (recombinant)cell, or cell-free expression system, in which it is present, andpolynucleotides produced in such cells or cell-free systems which aresubsequently purified away from at least some other components. Theexogenous polynucleotide (nucleic acid) can be a contiguous stretch ofnucleotides existing in nature, or comprise two or more contiguousstretches of nucleotides from different sources (naturally occurringand/or synthetic) joined to form a single polynucleotide. Typically suchchimeric polynucleotides comprise at least an open reading frameencoding a polypeptide of the invention operably linked to a promotersuitable of driving transcription of the open reading frame in a cell ofinterest.

The % identity of a polynucleotide is determined by GAP (Needleman andWunsch, 1970) analysis (GCG program) with a gap creation penalty=5, anda gap extension penalty=0.3. The query sequence is at least 3,000nucleotides in length, and the GAP analysis aligns the two sequencesover a region of at least 3,000 nucleotides. Even more preferably, thequery sequence is at least 3,750 nucleotides in length and the GAPanalysis aligns the two sequences over a region of at least 3,750nucleotides. Even more preferably, the query sequence is at least 4,500nucleotides in length and the GAP analysis aligns the two sequences overa region of at least 4,500 nucleotides. Even more preferably, the GAPanalysis aligns two sequences over their entire length.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 95%, more preferably at least96%, more preferably at least 97%, more preferably at least 98%, morepreferably at least 99%, more preferably at least 99.1%, more preferablyat least 99.2%, more preferably at least 99.3%, more preferably at least99.4%, more preferably at least 99.5%, more preferably at least 99.6%,more preferably at least 99.7%, more preferably at least 99.8%, and evenmore preferably at least 99.9% identical to the relevant nominated SEQID NO.

The present invention also relates to the use of oligonucleotides, forinstance in methods of screening for a polynucleotide of, or encoding apolypeptide of, the invention. As used herein, “oligonucleotides” arepolynucleotides up to 50 nucleotides in length. The minimum size of sucholigonucleotides is the size required for the formation of a stablehybrid between an oligonucleotide and a complementary sequence on anucleic acid molecule of the present invention. They can be RNA, DNA, orcombinations or derivatives of either. Oligonucleotides are typicallyrelatively short single stranded molecules of 10 to 30 nucleotides,commonly 15-25 nucleotides in length. When used as a probe or as aprimer in an amplification reaction, the minimum size of such anoligonucleotide is the size required for the formation of a stablehybrid between the oligonucleotide and a complementary sequence on atarget nucleic acid molecule. Preferably, the oligonucleotides are atleast 15 nucleotides, more preferably at least 18 nucleotides, morepreferably at least 19 nucleotides, more preferably at least 20nucleotides, even more preferably at least 25 nucleotides in length.Oligonucleotides of the present invention used as a probe are typicallyconjugated with a label such as a radioisotope, an enzyme, biotin, afluorescent molecule or a chemiluminescent molecule.

The present invention includes oligonucleotides that can be used as, forexample, probes to identify nucleic acid molecules, or primers toproduce nucleic acid molecules. Probes and/or primers can be used toclone homologues of the polynucleotides of the invention from otherspecies. Furthermore, hybridization techniques known in the art can alsobe used to screen genomic or cDNA libraries for such homologues.

Polynucleotides and oligonucleotides of the present invention includethose which hybridize under stringent conditions to one or more of thesequences provided as SEQ ID NO: 1 or 2. As used herein, stringentconditions are those that (1) employ low ionic strength and hightemperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% NaDodSO₄ at 50° C.; (2) employ during hybridisation adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone,50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1%SDS.

Polynucleotides of the present invention may possess, when compared tonaturally occurring molecules, one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagenesis on the nucleic acid). A variant of a polynucleotide or anoligonucleotide of the invention includes molecules of varying sizes of,and/or are capable of hybridising to, the rice genome close to that ofthe reference polynucleotide or oligonucleotide molecules definedherein, preferably the endogenous ROS1a gene. For example, variants maycomprise additional nucleotides (such as 1, 2, 3, 4, or more), or lessnucleotides as long as they still hybridise to the target region.Furthermore, a few nucleotides may be substituted without influencingthe ability of the oligonucleotide to hybridise to the target region. Inaddition, variants may readily be designed which hybridise close to, forexample to within 50 nucleotides, the region of the plant genome wherethe specific oligonucleotides defined herein hybridise. In particular,this includes polynucleotides which encode the same polypeptide or aminoacid sequence but which vary in nucleotide sequence by redundancy of thegenetic code. The terms “polynucleotide variant” and “variant” alsoinclude naturally occurring allelic variants.

Genetic Variations

As used herein, the term “genetic variation” refers to one or more cellsof the grain, preferably cells in at least one or more or all ofaleurone, pericarp, nucellar projection, ovary, testa and starchyendosperm of the developing grain, or of a plant or part thereof of theinvention which have a genetic modification which may be introduced byman, or may be naturally occurring in rice plant (for example, crossedto produce a plant of the invention).

As used herein, the term “one or more introduced genetic variations”refers to one or more cells of the grain, preferably cells in at leastone or more or all of aleurone, pericarp, nucellar projection, ovary,testa and starchy endosperm of the developing grain, or of a plant orpart thereof of the invention which have a genetic modificationintroduced by man. In a preferred embodiment, every cell in the grain orthe plant or part thereof comprises the introduced genetic variation. Asthe skilled person would understand, there are many different types ofgenetic modifications which can be made such as, but not limited to, anucleic construct encoding an exogenous polynucleotide which reduces theexpression of a ROS1a gene (such as a dsRNA molecule or microRNA), anucleic construct encoding an exogenous polynucleotide which encodes aROS1a polypeptide whose amino acid sequence is different to the aminoacid sequence of a corresponding wild-type ROS1a polypeptide and whichhas reduced (preferably some but can be no) DNA glycosylase activitywhen compared to the corresponding wild-type ROS1a polypeptide, thegenome manipulated by gene editing to reduce the activity of anendogenous ROS1a gene, and using TILLING to introduce mutations andselect for plants producing grain with reduced ROS1a polypeptide DNAglycosylase activity.

As used herein, the term “reduce the activity of at least one ROS1agene” as it relates to the “one or more genetic variations” or “one ormore introduced genetic variations” refers to the genetic variationresulting in a reduction in the amount or activity of a ROS1apolypeptide expressed by the gene when compared to a correspondingwild-type rice plant. In an embodiment, the grain comprises a ROS1apolypeptide with at least some DNA glycosylase activity.

In an embodiment, the genetic variation does not down-regulate the DNAglycosylase activity of a non-ROS1a polypeptide. For example, thegenetic variation does not reduce the DNA glycosylase activity of eachof the ROS1b, ROS1c and ROS1d polypeptides by more than 10% or 30% inthe rice plant of the invention. Alternatively, the genetic variationreduces the DNA glycosylase activity of at least one of the ROS1b, ROS1cand ROS1d polypeptides by at least 30%.

RNA Interference

RNA interference (RNAi) is particularly useful for specifically reducingthe expression of a gene, which results in reduced production of aparticular protein if the gene encodes a protein. Although not wishingto be limited by theory, Waterhouse et al. (1998) have provided a modelfor the mechanism by which dsRNA (duplex RNA) can be used to reduceprotein production. This technology relies on the presence of dsRNAmolecules that contain a sequence that is essentially identical to themRNA of the gene of interest or part thereof. Conveniently, the dsRNAcan be produced from a single promoter in a recombinant vector or hostcell, where the sense and anti-sense sequences are flanked by anunrelated sequence which enables the sense and anti-sense sequences tohybridize to form the dsRNA molecule with the unrelated sequence forminga loop structure. The design and production of suitable dsRNA moleculesis well within the capacity of a person skilled in the art, particularlyconsidering Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619,WO 99/53050, WO 99/49029 and WO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology to a ROS1agene. The DNA therefore comprises both sense and antisense sequencesthat, when transcribed into RNA, can hybridize to form the doublestranded RNA region. In one embodiment of the invention, the sense andantisense sequences are separated by a spacer region that comprises anintron which, when transcribed into RNA, is spliced out. Thisarrangement has been shown to result in a higher efficiency of genesilencing (Smith et al., 2000). The double stranded region may compriseone or two RNA molecules, transcribed from either one DNA region or two.The presence of the double stranded molecule is thought to trigger aresponse from an endogenous system that destroys both the doublestranded RNA and also the homologous RNA transcript from the targetgene, efficiently reducing or eliminating the activity of the targetgene.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides, preferably at least 30 or atleast 50 contiguous nucleotides, more preferably at least 100 or atleast 200 contiguous nucleotides. Generally, a sequence of 100-1000nucleotides corresponding to a region of the target gene mRNA is used.The full-length sequence corresponding to the entire gene transcript maybe used. The degree of identity of the sense sequence to the targetedtranscript (and therefore also the identity of the antisense sequence tothe complement of the target transcript) should be at least 85%, atleast 90%, or 95-100%, preferably is identical to the targeted sequence.The RNA molecule may of course comprise unrelated sequences which mayfunction to stabilize the molecule. The RNA molecule may be expressedunder the control of a RNA polymerase II or RNA polymerase III promoter.Examples of the latter include tRNA or snRNA promoters.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-25 contiguousnucleotides of the target mRNA. Preferably, the siRNA sequence commenceswith the dinucleotide AA, comprises a GC-content of about 30-70%(preferably, 30-60%, more preferably 40-60% and more preferably about45%-55%), and does not have a high percentage identity to any nucleotidesequence other than the target in the genome of the organism in which itis to be introduced, for example, as determined by standard BLASTsearch.

DsRNA's useful for the invention could readily be produced using routineprocedures.

microRNA

MicroRNAs (abbreviated miRNAs) are non-coding RNA molecules having alength generally 19-25 nucleotides (commonly about 20-24 nucleotides inplants) that are derived from larger precursors that form imperfectstem-loop structures. The miRNA is typically fully complementary to aregion of a target mRNA whose expression is to be reduced, but need notbe fully complementary.

miRNAs bind to complementary sequences on target messenger RNAtranscripts (mRNAs), usually resulting in translational repression ortarget degradation and gene silencing. Artificial miRNAs (amiRNAs) canbe designed based on natural miRNAs for reducing the expression of anygene of interest, as well known in the art.

In plant cells, miRNA precursor molecules are believed to be largelyprocessed in the nucleus. The pri-miRNA (containing one or more localdouble-stranded or “hairpin” regions as well as the usual 5′ “cap” andpolyadenylated tail of an mRNA) is processed to a shorter miRNAprecursor molecule that also includes a stem-loop or fold-back structureand is termed the “pre-miRNA”. In plants, the pre-miRNAs are cleaved bydistinct DICER-like (DCL) enzymes, yielding miRNA:miRNA* duplexes. Priorto transport out of the nucleus, these duplexes are methylated.

In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex isselectively incorporated into an active RNA-induced silencing complex(RISC) for target recognition. The RISC-complexes contain a particularsubset of Argonaute proteins that exert sequence-specific generepression (see, for example, Millar and Waterhouse, 2005; Pasquinelliet al., 2005; Almeida and Allshire, 2005).

MicroRNA's useful for the invention could readily be produced usingroutine procedures. For example, the design of a ROS1a amiRNA(artificial microRNA) construct may be based on the general methoddescribed by Fahim et al. (2012). WMD3 software(www.wmd3.weigelworld.org/) can be used to identify suitable amiRNAtargets in a ROS1a gene. The amiRNA targets are selected according tofour criteria: 1) relative 5′ instability by using sequences which areAT rich at the 5′-end and GC rich at the 3′-end; 2) U at position 1 andA at the cleavage site (between positions 10 and 11); 3) maximum of 1and 4 mismatches at positions 1 to 9, and 13 to 21, respectively; and 4)having a predicted free energy (AG) of less than −30 kcal mol⁻¹ when theamiRNA would hybridise to the target RNA (Ossowski et. al., 2008). Forgene-specific reduction of expression, candidate amiRNA sequences arechosen in a region which shows the lowest homology upon the alignment ofall the homologs of OsROS1a, thus reducing the potential for off-targetreduction of the expression of ROS1 homologs and homoeologs. Theprecursor of rice miR395 (Guddeti et al., 2005; Jones-Rhoades andBartel, 2004; Kawashima et al., 2009) may be chosen as the amiRNAbackbone for insertion of the amiRNA sequences. To design and make theconstruct, five endogenous miRNA targets in the miR395 were replaced byfive amiRNA targets for TA2 knock down.

Cosuppression

Genes can suppress the expression of related endogenous genes and/ortransgenes already present in the genome, a phenomenon termedhomology-dependent gene silencing. Most of the instances of homologydependent gene silencing fall into two classes—those that function atthe level of transcription of the transgene, and those that operatepost-transcriptionally.

Post-transcriptional homology-dependent gene silencing (i.e.,cosuppression) describes the loss of expression of a transgene andrelated endogenous or viral genes in transgenic plants. Cosuppressionoften, but not always, occurs when transgene transcripts are abundant,and it is generally thought to be triggered at the level of mRNAprocessing, localization, and/or degradation. Several models exist toexplain how cosuppression works (see in Taylor, 1997).

Cosuppression involves introducing an extra copy of a gene or a fragmentthereof into a plant in the sense orientation with respect to a promoterfor its expression. The size of the sense fragment, its correspondenceto target gene regions, and its degree of sequence identity to thetarget gene can be determined by those skilled in the art. In someinstances, the additional copy of the gene sequence interferes with theexpression of the target plant gene. Reference is made to WO 97/20936and EP 0465572 for methods of implementing co-suppression approaches.

Antisense Polynucleotides

The term “antisense polynucleotide” shall be taken to mean a DNA or RNAmolecule that is complementary to at least a portion of a specific mRNAmolecule encoding an endogenous polypeptide and capable of interferingwith a post-transcriptional event such as mRNA translation. The use ofantisense methods is well known in the art (see for example, G. Hartmannand S. Endres, Manual of Antisense Methodology, Kluwer (1999)). The useof antisense techniques in plants has been reviewed by Bourque (1995)and Senior (1998). Bourque (1995) lists a large number of examples ofhow antisense sequences have been utilized in plant systems as a methodof gene inactivation. Bourque also states that attaining 100% inhibitionof any enzyme activity may not be necessary as partial inhibition willmore than likely result in measurable change in the system. Senior(1998) states that antisense methods are now a very well establishedtechnique for manipulating gene expression.

In one embodiment, the antisense polynucleotide hybridises underphysiological conditions, that is, the antisense polynucleotide (whichis fully or partially single stranded) is at least capable of forming adouble stranded polynucleotide with mRNA encoding an endogenous ROS1apolypeptide under normal conditions in a cell.

Antisense molecules may include sequences that correspond to thestructural genes or for sequences that effect control over the geneexpression or splicing event. For example, the antisense sequence maycorrespond to the targeted coding region of endogenous gene, or the5′-untranslated region (UTR) or the 3′-UTR or combination of these. Itmay be complementary in part to intron sequences, which may be splicedout during or after transcription, preferably only to exon sequences ofthe target gene. In view of the generally greater divergence of theUTRs, targeting these regions provides greater specificity of geneinhibition.

The length of the antisense sequence should be at least 19 contiguousnucleotides, preferably at least 30 or at least 50 nucleotides, and morepreferably at least 100, 200, 500 or 1000 nucleotides. The full-lengthsequence complementary to the entire gene transcript may be used. Thelength is most preferably 100-2000 nucleotides. The degree of identityof the antisense sequence to the targeted transcript should be at least90% and more preferably 95-100%, typically 100% identical. The antisenseRNA molecule may of course comprise unrelated sequences which mayfunction to stabilize the molecule.

Genome Editing Using Site-Specific Nucleases

Genome editing uses engineered nucleases composed of sequence specificDNA binding domains fused to a non-specific DNA cleavage module. Thesechimeric nucleases enable efficient and precise genetic modifications byinducing targeted DNA double stranded breaks that stimulate the cell'sendogenous cellular DNA repair mechanisms to repair the induced break.Such mechanisms include, for example, error prone non-homologous endjoining (NHEJ) and homology directed repair (HDR).

In the presence of donor plasmid with extended homology arms, HDR canlead to the introduction of single or multiple transgenes to correct orreplace existing genes. In the absence of donor plasmid, NHEJ-mediatedrepair yields small insertion or deletion mutations of the target thatcause gene disruption.

Engineered nucleases useful in the methods of the present inventioninclude zinc finger nucleases (ZFNs), transcription activator-like (TAL)effector nucleases (TALEN) and CRISPR-Cas9 type site-specific nucleases.

Typically nuclease encoded genes are delivered into cells by plasmidDNA, viral vectors or in vitro transcribed mRNA. The use of fluorescentsurrogate reporter vectors also allows for enrichment of ZFN-, TALEN- orCRISPR-modified cells.

Complex genomes often contain multiple copies of sequences that areidentical or highly homologous to the intended DNA target, potentiallyleading to off-target activity and cellular toxicity. To address this,structure (Miller et al., 2007; Szczepek et al., 2007) and selectionbased (Doyon et al., 2011; Guo et al., 2010) approaches can be used togenerate improved ZFN and TALEN heterodimers with optimized cleavagespecificity and reduced toxicity.

In order to target genetic recombination or mutation by ZFN according toa preferred embodiment of the present invention, two 9 bp zinc fingerDNA recognition sequences must be identified in the host DNA. Theserecognition sites will be in an inverted orientation with respect to oneanother and separated by about 6 bp of DNA. ZFNs are then generated bydesigning and producing zinc finger combinations that bind DNAspecifically at the target locus, and then linking the zinc fingers to aDNA cleavage domain.

A transcription activator-like (TAL) effector nuclease (TALEN) comprisesa TAL effector DNA binding domain and an endonuclease domain.

TAL effectors are proteins of plant pathogenic bacteria that areinjected by the pathogen into the plant cell, where they travel to thenucleus and function as transcription factors to turn on specific plantgenes. The primary amino acid sequence of a TAL effector dictates thenucleotide sequence to which it binds. Thus, target sites can bepredicted for TAL effectors, and TAL effectors can be engineered andgenerated for the purpose of binding to particular nucleotide sequences.

Fused to the TAL effector-encoding nucleic acid sequences are sequencesencoding a nuclease or a portion of a nuclease, typically a nonspecificcleavage domain from a type II restriction endonuclease such as FokI(Kim et al., 1996). Other useful endonucleases may include, for example,HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. The fact that someendonucleases (e.g., FokI) only function as dimers can be capitalizedupon to enhance the target specificity of the TAL effector. For example,in some cases each FokI monomer can be fused to a TAL effector sequencethat recognizes a different DNA target sequence, and only when the tworecognition sites are in close proximity do the inactive monomers cometogether to create a functional enzyme. By requiring DNA binding toactivate the nuclease, a highly site-specific restriction enzyme can becreated.

A sequence-specific TALEN can recognize a particular sequence within apreselected target nucleotide sequence present in a cell. Thus, in someembodiments, a target nucleotide sequence can be scanned for nucleaserecognition sites, and a particular nuclease can be selected based onthe target sequence. In other cases, a TALEN can be engineered to targeta particular cellular sequence.

Nucleic Acid Constructs

The present invention includes nucleic acid constructs comprising thepolynucleotides of or useful for the invention, and vectors and hostcells containing these, methods of their production and use, and usesthereof.

The present invention refers to elements which are operably connected orlinked. “Operably connected” or “operably linked” and the like refer toa linkage of polynucleotide elements in a functional relationship.Typically, operably connected nucleic acid sequences are contiguouslylinked and, where necessary to join two protein coding regions,contiguous and in reading frame. A coding sequence is “operablyconnected to” another coding sequence when RNA polymerase willtranscribe the two coding sequences into a single RNA, which iftranslated is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

As used herein, the term “cis-acting sequence”, “cis-acting element” or“cis-regulatory region” or “regulatory region” or similar term shall betaken to mean any sequence of nucleotides, which when positionedappropriately and connected relative to an expressible genetic sequence,is capable of regulating, at least in part, the expression of thegenetic sequence. Those skilled in the art will be aware that acis-regulatory region may be capable of activating, silencing,enhancing, repressing or otherwise altering the level of expressionand/or cell-type-specificity and/or developmental specificity of a genesequence at the transcriptional or post-transcriptional level. Inpreferred embodiments of the present invention, the cis-acting sequenceis an activator sequence that enhances or stimulates the expression ofan expressible genetic sequence.

“Operably connecting” a promoter or enhancer element to a transcribablepolynucleotide means placing the transcribable polynucleotide (e.g.,protein-encoding polynucleotide or other transcript) under theregulatory control of a promoter, which then controls the transcriptionof that polynucleotide. In the construction of heterologouspromoter/structural gene combinations, it is generally preferred toposition a promoter or variant thereof at a distance from thetranscription start site of the transcribable polynucleotide which isapproximately the same as the distance between that promoter and theprotein coding region it controls in its natural setting; i.e., the genefrom which the promoter is derived. As is known in the art, somevariation in this distance can be accommodated without loss of function.Similarly, the preferred positioning of a regulatory sequence element(e.g., an operator, enhancer etc) with respect to a transcribablepolynucleotide to be placed under its control is defined by thepositioning of the element in its natural setting; i.e., the genes fromwhich it is derived.

“Promoter” or “promoter sequence” as used herein refers to a region of agene, generally upstream (5′) of the RNA encoding region, which controlsthe initiation and level of transcription in the cell of interest. A“promoter” includes the transcriptional regulatory sequences of aclassical genomic gene, such as a TATA box and CCAAT box sequences, aswell as additional regulatory elements (i.e., upstream activatingsequences, enhancers and silencers) that alter gene expression inresponse to developmental and/or environmental stimuli, or in atissue-specific or cell-type-specific manner. A promoter is usually, butnot necessarily (for example, some PolIII promoters), positionedupstream of a structural gene, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.Promoters may contain additional specific regulatory elements, locatedmore distal to the start site to further enhance expression in a cell,and/or to alter the timing or inducibility of expression of a structuralgene to which it is operably connected.

“Constitutive promoter” refers to a promoter that directs expression ofan operably linked transcribed sequence in many or all tissues of anorganism such as a plant. The term constitutive as used herein does notnecessarily indicate that a gene is expressed at the same level in allcell types, but that the gene is expressed in a wide range of celltypes, although some variation in level is often detectable. “Selectiveexpression” as used herein refers to expression almost exclusively inspecific organs of, for example, the plant, such as, for example,endosperm, embryo, leaves, fruit, tubers or root. In a preferredembodiment, a promoter is expressed selectively or preferentially ingrain of a plant, preferably a rice plant. Selective expression maytherefore be contrasted with constitutive expression, which refers toexpression in many or all tissues of a plant under most or all of theconditions experienced by the plant.

Selective expression may also result in compartmentation of the productsof gene expression in specific plant tissues, organs or developmentalstages. Compartmentation in specific subcellular locations such as theplastid, cytosol, vacuole, or apoplastic space may be achieved by theinclusion in the structure of the gene product of appropriate signals,eg. a signal peptide, for transport to the required cellularcompartment, or in the case of the semi-autonomous organelles (plastidsand mitochondria) by integration of the transgene with appropriateregulatory sequences directly into the organelle genome.

A “tissue-specific promoter” or “organ-specific promoter” is a promoterthat is preferentially expressed in one tissue or organ relative to manyother tissues or organs, preferably most if not all other tissues ororgans in, for example, a plant. Typically, the promoter is expressed ata level 10-fold higher in the specific tissue or organ than in othertissues or organs.

Seed specific promoters for the invention which are suitable are theoilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Viciafaba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosinpromoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (U.S.Pat. No. 5,504,200), the Brassica Bce4 promoter (WO 91/13980) or thelegumin B4 promoter (Baumlein et al., 1992), and promoters which lead tothe seed-specific expression in rice and the like. Notable promoterswhich are suitable are the barley LPT2 or LPT1 gene promoters (WO95/15389 and WO 95/23230) or the promoters described in WO 99/16890(promoters from the barley hordein gene). Other promoters include thosedescribed by Broun et al. (1998), Potenza et al. (2004), US 20070192902and US 20030159173. In an embodiment, the seed specific promoter ispreferentially expressed in defined parts of the seed such as theendosperm, preferably the developing aleurone. In a further embodiment,the seed specific promoter is not expressed, or is only expressed at alow level, after the seed germinates.

In an embodiment, the promoter is at least active at a time pointbetween the time of anthesis and 7 days post-anthesis, or activeentirely during this period. An example of such a promoter is a ROS1agene promoter.

In an embodiment, the promoter operably linked to an exogenouspolynucleotide which reduces the expression of a ROS1a gene is not ahigh MW glutenin promoter.

The promoters contemplated by the present invention may be native to thehost plant to be transformed or may be derived from an alternativesource, where the region is functional in the host plant. Other sourcesinclude the Agrobacterium T-DNA genes, such as the promoters of genesfor the biosynthesis of nopaline, octapine, mannopine, or other opinepromoters, tissue specific promoters (see, e.g., U.S. Pat. No. 5,459,252and WO 91/13992); promoters from viruses (including host specificviruses), or partially or wholly synthetic promoters. Numerous promotersthat are functional in mono- and dicotyledonous plants are well known inthe art (see, for example, Greve, 1983; Salomon et al., 1984; Garfinkelet al., 1983; Barker et al., 1983); including various promoters isolatedfrom plants and viruses such as the cauliflower mosaic virus promoter(CaMV 35S, 19S). Non-limiting methods for assessing promoter activityare disclosed by Medberry et al. (1992 and 1993), Sambrook et al. (1989,supra) and U.S. Pat. No. 5,164,316.

Alternatively or additionally, the promoter may be an inducible promoteror a developmentally regulated promoter which is capable of drivingexpression of the introduced polynucleotide at an appropriatedevelopmental stage of the, for example, plant. Other cis-actingsequences which may be employed include transcriptional and/ortranslational enhancers. Enhancer regions are well known to personsskilled in the art, and can include an ATG translational initiationcodon and adjacent sequences. When included, the initiation codon shouldbe in phase with the reading frame of the coding sequence relating tothe foreign or exogenous polynucleotide to ensure translation of theentire sequence if it is to be translated. Translational initiationregions may be provided from the source of the transcriptionalinitiation region, or from a foreign or exogenous polynucleotide. Thesequence can also be derived from the source of the promoter selected todrive transcription, and can be specifically modified so as to increasetranslation of the mRNA.

The nucleic acid construct of the present invention may comprise a3′-non-translated sequence from about 50 to 1,000 nucleotide base pairswhich may include a transcription termination sequence. A 3′non-translated sequence may contain a transcription termination signalwhich may or may not include a polyadenylation signal and any otherregulatory signals capable of effecting mRNA processing. Apolyadenylation signal functions for addition of polyadenylic acidtracts to the 3′ end of a mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form 5′AATAAA-3′ although variations are not uncommon. Transcriptiontermination sequences which do not include a polyadenylation signalinclude terminators for Poll or PolIII RNA polymerase which comprise arun of four or more thymidines. Examples of suitable 3′ non-translatedsequences are the 3′ transcribed non-translated regions containing apolyadenylation signal from an octopine synthase (ocs) gene or nopalinesynthase (nos) gene of Agrobacterium tumefaciens (Bevan et al., 1983).Suitable 3′ non-translated sequences may also be derived from plantgenes such as the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO)gene, although other 3′ elements known to those of skill in the art canalso be employed.

As the DNA sequence inserted between the transcription initiation siteand the start of the coding sequence, i.e., the untranslated 5′ leadersequence (5′UTR), can influence gene expression if it is translated aswell as transcribed, one can also employ a particular leader sequence.Suitable leader sequences include those that comprise sequences selectedto direct optimum expression of the foreign or endogenous DNA sequence.For example, such leader sequences include a preferred consensussequence which can increase or maintain mRNA stability and preventinappropriate initiation of translation as for example described byJoshi (1987).

Vectors

The present invention includes use of vectors for manipulation ortransfer of genetic constructs. By “chimeric vector” is meant a nucleicacid molecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, or plant virus, into which a nucleic acidsequence may be inserted or cloned. A vector preferably isdouble-stranded DNA and contains one or more unique restriction sitesand may be capable of autonomous replication in a defined host cellincluding a target cell or tissue or a progenitor cell or tissuethereof, or capable of integration into the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectormay be an autonomously replicating vector, i.e., a vector that exists asan extrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into a cell,is integrated into the genome of the recipient cell and replicatedtogether with the chromosome(s) into which it has been integrated. Avector system may comprise a single vector or plasmid, two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the cell into which the vector is to be introduced. The vector mayalso include a selection marker such as an antibiotic resistance gene, aherbicide resistance gene or other gene that can be used for selectionof suitable transformants. Examples of such genes are well known tothose of skill in the art.

The nucleic acid construct of the invention can be introduced into avector, such as a plasmid. Plasmid vectors typically include additionalnucleic acid sequences that provide for easy selection, amplification,and transformation of the expression cassette in prokaryotic andeukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, orbinary vectors containing one or more T-DNA regions. Additional nucleicacid sequences include origins of replication to provide for autonomousreplication of the vector, selectable marker genes, preferably encodingantibiotic or herbicide resistance, unique multiple cloning sitesproviding for multiple sites to insert nucleic acid sequences or genesencoded in the nucleic acid construct, and sequences that enhancetransformation of prokaryotic and eukaryotic (especially plant) cells.

By “marker gene” is meant a gene that imparts a distinct phenotype tocells expressing the marker gene and thus allows such transformed cellsto be distinguished from cells that do not have the marker. A selectablemarker gene confers a trait for which one can “select” based onresistance to a selective agent (e.g., a herbicide, antibiotic,radiation, heat, or other treatment damaging to untransformed cells). Ascreenable marker gene (or reporter gene) confers a trait that one canidentify through observation or testing, i.e., by “screening” (e.g.,β-glucuronidase, luciferase, GFP or other enzyme activity not present inuntransformed cells). The marker gene and the nucleotide sequence ofinterest do not have to be linked.

To facilitate identification of transformants, the nucleic acidconstruct desirably comprises a selectable or screenable marker gene as,or in addition to, the foreign or exogenous polynucleotide. The actualchoice of a marker is not crucial as long as it is functional (i.e.,selective) in combination with the plant cells of choice. The markergene and the foreign or exogenous polynucleotide of interest do not haveto be linked, since co-transformation of unlinked genes as, for example,described in U.S. Pat. No. 4,399,216 is also an efficient process inplant transformation.

Examples of bacterial selectable markers are markers that conferantibiotic resistance such as ampicillin, erythromycin, chloramphenicolor tetracycline resistance, preferably kanamycin resistance. Exemplaryselectable markers for selection of plant transformants include, but arenot limited to, a hyg gene which encodes hygromycin B resistance; aneomycin phosphotransferase (nptII) gene conferring resistance tokanamycin, paromomycin, G418; a glutathione-S-transferase gene from ratliver conferring resistance to glutathione derived herbicides as, forexample, described in EP 256223; a glutamine synthetase gene conferring,upon overexpression, resistance to glutamine synthetase inhibitors suchas phosphinothricin as, for example, described in WO 87/05327, anacetyltransferase gene from Streptomyces viridochromogenes conferringresistance to the selective agent phosphinothricin as, for example,described in EP 275957, a gene encoding a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) conferring toleranceto N-phosphonomethylglycine as, for example, described by Hinchee et al.(1988), a bar gene conferring resistance against bialaphos as, forexample, described in WO91/02071; a nitrilase gene such as bxn fromKlebsiella ozaenae which confers resistance to bromoxynil (Stalker etal., 1988); a dihydrofolate reductase (DHFR) gene conferring resistanceto methotrexate (Thillet et al., 1988); a mutant acetolactate synthasegene (ALS), which confers resistance to imidazolinone, sulfonylurea orother ALS-inhibiting chemicals (EP 154,204); a mutated anthranilatesynthase gene that confers resistance to 5-methyl tryptophan; or adalapon dehalogenase gene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known, a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known, an aequorin gene(Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof, a luciferase (luc) gene (Ow et al.,1986), which allows for bioluminescence detection, and others known inthe art. By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that facilitates determination of promoter activityby reference to protein product.

Preferably, the nucleic acid construct is stably incorporated into thegenome of, for example, the plant or cell of the invention. Accordingly,the nucleic acid comprises appropriate elements which allow the moleculeto be incorporated into the genome, or the construct is placed in anappropriate vector which can be incorporated into a chromosome of aplant cell.

One embodiment of the present invention includes a recombinant vector,which includes at least one polynucleotide molecule of the presentinvention, inserted into any vector capable of delivering the nucleicacid molecule into a host cell. Such a vector contains heterologousnucleic acid sequences, that is nucleic acid sequences that are notnaturally found adjacent to nucleic acid molecules of the presentinvention and that preferably are derived from a species other than thespecies from which the nucleic acid molecule(s) are derived. The vectorcan be either RNA or DNA, either prokaryotic or eukaryotic, andtypically is a virus or a plasmid.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

The level of a ROS1s polypeptide may be modulated by decreasing thelevel of expression of a gene encoding the protein in the rice plant,leading to increased aleurone thickness. The level of expression of agene may be modulated by altering the copy number per cell, for exampleby introducing a synthetic genetic construct comprising the codingsequence and a transcriptional control element that is operablyconnected thereto and that is functional in the cell. A plurality oftransformants may be selected and screened for those with a favourablelevel and/or specificity of transgene expression arising from influencesof endogenous sequences in the vicinity of the transgene integrationsite. A favourable level and pattern of transgene expression is onewhich results in increased aleurone thickness. Alternatively, apopulation of mutagenized seed or a population of plants from a breedingprogram may be screened for individual lines with increased aleuronethickness.

Recombinant Cells

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention, or progeny cells thereof.Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. Transformed nucleic acid molecules of the present inventioncan remain extrachromosomal or can integrate into one or more siteswithin a chromosome of the transformed (i.e., recombinant) cell in sucha manner that their ability to be expressed is retained. Preferred hostcells are plant cells, more preferably a rice cell.

Plants with Genetic Variations

The term “plant” as used herein as a noun refers to whole plants andrefers to any member of the Kingdom Plantae, but as used as an adjectiverefers to any substance which is present in, obtained from, derivedfrom, or related to a plant, such as for example, plant organs (e.g.leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plantcells and the like. Plantlets and germinated seeds from which roots andshoots have emerged are also included within the meaning of “plant”. Theterm “plant parts” as used herein refers to one or more plant tissues ororgans which are obtained from a plant and which comprises genomic DNAof the plant. Plant parts include vegetative structures (for example,leaves, stems), roots, floral organs/structures, seed (including embryo,cotyledons, and seed coat), plant tissue (for example, vascular tissue,ground tissue, and the like), cells and progeny of the same. The term“plant cell” as used herein refers to a cell obtained from a plant or ina plant and includes protoplasts or other cells derived from plants,gamete-producing cells, and cells which regenerate into whole plants.Plant cells may be cells in culture. By “plant tissue” is meantdifferentiated tissue in a plant or obtained from a plant (“explant”) orundifferentiated tissue derived from immature or mature embryos, seeds,roots, shoots, fruits, tubers, pollen, tumor tissue, such as crowngalls, and various forms of aggregations of plant cells in culture, suchas calli. Exemplary plant tissues in or from seeds are cotyledon, embryoand embryo axis. The invention accordingly includes plants and plantparts and products comprising these.

The terms “grain” and “seed” are used interchangeably herein. “Grain”can refer to mature grain in the plant, developing grain in the plant,harvested grain or to grain after processing such as, for example,milling or polishing, where most of the grain stays intact, or afterimbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18-20%. In anembodiment, developing grain of the invention is at least about 10 daysafter pollination (DAP). In an embodiment, developing grain of theinvention is at least includes grain between anthesis and 7 dayspost-anthesis.

A “transgenic plant” as used herein refers to a plant with one or moregenetic variations as defined herein such contains a nucleic acidconstruct not found in a wild-type plant of the same species, variety orcultivar. That is, transgenic plants (transformed plants) containgenetic material (a transgene) that they did not contain prior to thetransformation. The transgene may include genetic sequences obtainedfrom or derived from a plant cell, or another plant cell, or a non-plantsource, or a synthetic sequence. Typically, the transgene has beenintroduced into the plant by human manipulation such as, for example, bytransformation but any method can be used as one of skill in the artrecognizes. The genetic material is preferably stably integrated intothe genome of the plant. The introduced genetic material may comprisesequences that naturally occur in the same species but in a rearrangedorder or in a different arrangement of elements, for example anantisense sequence. Plants containing such sequences are included hereinin “transgenic plants”.

A “non-transgenic plant” is one which has not been genetically modifiedby the introduction of genetic material by recombinant DNA techniques.

“Wild-type”, as used herein, refers to a cell, tissue, grain or plantthat has not been modified according to the invention. Wild-type cells,tissue or plants may be used as controls to compare levels of expressionof an exogenous nucleic acid or the extent and nature of traitmodification with cells, tissue, grain or plants modified as describedherein.

As used herein, the term “corresponding wild-type” rice plant or grain,or similar phrases, refers to a rice plant or grain which comprises atleast 50%, more preferably at least 75%, more preferably at least 95%,more preferably at least 97%, more preferably at least 99%, and evenmore preferably 99.5% of the genotype of a rice plant or grain of theinvention, but does not comprise the one or more genetic variations(such as introduced genetic variations) which each reduce the activityof a ROS1a gene in the plant or grain, and/or a thickened aleurone. Inan embodiment, a rice grain or plant of the invention is isogenicrelative a wild-type rice grain or plant apart from the one or moregenetic variations (such as introduced genetic variations). Preferably,the corresponding wild-type plant or grain is of/from the same cultivaror variety as the progenitor of the plant/grain of the invention, or asibling plant line which lacks the one or more genetic modificationsand/or does not have a thickened aleurone, often termed a “segregant”.In an embodiment, the rice plant or grain of the invention has agenotype that is less than 50% identical to the genotype of ricecultivar Zhonghua 11 (ZH11). ZH11 has been commercially available since1986.

Transgenic plants, as defined in the context of the present inventioninclude progeny of the rice plants which have been genetically modifiedusing recombinant techniques, wherein the progeny comprise the transgeneof interest. Such progeny may be obtained by self-fertilisation of theprimary transgenic plant or by crossing such plants with another riceplant. This would generally be to modulate the production of at leastone protein defined herein in the desired plant or plant organ.Transgenic plant parts include all parts and cells of said plantscomprising the transgene such as, for example, cultured tissues, callusand protoplasts.

As used herein, the term “rice” refers to any species of the GenusOryza, including progenitors thereof, as well as progeny thereofproduced by crosses with other species. It is preferred that the plantis of a Oryza species which is commercially cultivated such as, forexample, a strain or cultivar or variety of Oryza sativa or suitable forcommercial production of grain.

As used herein, “brown rice” means the whole grain of rice including thebran layer and embryo (germ) but not the hull which has been removed,usually during harvesting. That is, brown rice has not been polished toremove the aleurone and embryo. The “brown” refers to the presence ofbrown or yellow-brown pigments in the bran layer. Brown rice isconsidered a wholegrain. As used herein “white rice” (milled rice) meansrice grain from which the bran and germ have been removed i.e.essentially the starchy endosperm of the whole rice grain. Both of theseclasses of rice grain may come in short, medium or long grain forms.Compared with white rice, brown rice has a higher content of protein,minerals and vitamins and a higher lysine content in its proteincontent.

As used herein, “pigmented rice” includes black rice and red rice, eachof which contain pigments in the aleurone layer, such asproanthocyanidins (tannins). Pigmented rice has a higher riboflavincontent than non-pigmented rice, but similar thimine content. “Blackrice” has a black or almost black coloured bran layer due toanthocyanins, and may turn a deep purple colour upon cooking. “Purplerice” (also known as “forbidden rice”) is a short grain variant of blackrice and is included in black rice as defined here. It is purple incolour in the uncooked state and deep purple when cooked. “Red rice”contains a variety of anthocyanins that gives the bran a red/marooncolour, including cyanidin-3-glucoside (chrysanthemin) andpeonidin-3-glucoside (oxycoccicy-anin).

Each of these types of rice grain may be treated so as preventgermination, for example by cooking (boiling) or by dry heating. Brownand pigmented rice is typically cooked for 20-40 min, depending on thedesired texture, whereas white rice is typically cooked for 12-18 min.Cooking or heating reduces the levels of antinutritional factors in ricegrain such as trypsin inhibitor, oryzacystatin and haemagglutinins(lectins) by denaturation of these proteins, but not of the phytatecontent. Rice grain may also be soaked in water before cooking, orslow-cooked for longer times, as known in the art. Rice grain may alsobe cracked, parboiled, or heat-stabilised. Rice bran may be steamtreated to stabilise it, for example for about 6 min at 100° C.

In an embodiment, grain of the invention has delayed grain maturationwhen compared to corresponding wild-type grain. Delayed maturation canbe determined by using the seed setting rate (%) which entailscalculating the percentage of florets in the plant that were filled by aseed by the mature grain stage.

In an embodiment, grain of the invention has a decreased germinationcapacity when compared corresponding wild-type grain. For example, thegrain has about 70% to about 80%, or about 75%, preferably 70% to 100%,of the germination capacity of corresponding wild-type grain whencultured at 28° C. under 12 h light/12 h dark cycles without humiditycontrol in a growth chamber. The term “germination” as used herein isdefined as when the radicle had visibly emerged through the seed coat.

In an embodiment, plants of the invention have one or more or all ofnormal plant height, fertility (male and female), grain size and 1000grain weight relative to the wild-type parental variety (such as anisogenic plant comprising a ROS1a polypeptide with a sequence of aminoacids provided as SEQ ID NO: 2). In an embodiment, grain of theinvention is capable of producing a rice plant which has one or more orall of: normal plant height, fertility (male and female), grain size and1000 grain weight relative to the wild-type parental variety. As usedherein, the term “normal” can be determined by measuring the same traitin the wild-type parental variety grown under the same conditions as aplant of the invention. In an embodiment, to be normal a plant of theinvention has +/−10%, more preferably +/−5%, more preferably +/−2.5%,even more preferably +/−1%, of the level/number etc of the definedfeature when compared to the wild-type parental variety.

Transgenic plants, as defined in the context of the present inventioninclude plants (as well as parts and cells of said plants) and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide of thepresent invention in the desired plant or plant organ. Transgenic plantscan be produced using techniques known in the art, such as thosegenerally described in A. Slater et al., Plant Biotechnology—The GeneticManipulation of Plants, Oxford University Press (2003), and P. Christouand H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons(2004).

In a preferred embodiment, the transgenic plants are homozygous for eachand every gene or nucleic acid construct that has been introduced(transgene) so that their progeny do not segregate for the desiredphenotype. The transgenic plants may also be heterozygous for theintroduced transgene(s), such as, for example, in F1 progeny which havebeen grown from hybrid seed. Such plants may provide advantages such ashybrid vigour, well known in the art.

In an embodiment, a method of selecting a rice plant of the inventionfurther comprises analysing a DNA sample from the plant for at least one“other genetic marker”. As used herein, the “other genetic marker” maybe any molecules which are linked to a desired trait of a plant. Suchmarkers are well known to those skilled in the art and include molecularmarkers linked to genes determining traits such disease resistance,yield, plant morphology, grain quality, dormancy traits, grain colour,gibberellic acid content in the seed, plant height, flour colour and thelike. Examples of such genes are the Rht genes that determine asemi-dwarf growth habit and therefore lodging resistance.

Four general methods for direct delivery of a gene into cells have beendescribed: (1) chemical methods (Graham et al., 1973); (2) physicalmethods such as microinjection (Capecchi, 1980); electroporation (see,for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO92/09696 and WO 93/21335); and the gene gun (see, for example, U.S. Pat.Nos. 4,945,050 and 5,141,131); (3) viral vectors (Clapp, 1993; Lu etal., 1993; Eglitis et al., 1988); and (4) receptor-mediated mechanisms(Curiel et al., 1992; Wagner et al., 1992).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. A particle delivery system suitable for use with the presentinvention is the helium acceleration PDS-1000/He gun is available fromBio-Rad Laboratories. For the bombardment, immature embryos or derivedtarget cells such as scutella or calli from immature embryos may bearranged on solid culture medium.

In another alternative embodiment, plastids can be stably transformed.Method disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402,5,932,479, and WO 99/05265.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see, for example, U.S. Pat. Nos. 5,177,010, 5,104,310,5,004,863, 5,159,135). Further, the integration of the T-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants for the gene of interest.

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., Methods for Plant MolecularBiology, Academic Press, San Diego, (1988)). This regeneration andgrowth process typically includes the steps of selection of transformedcells, culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredexogenous nucleic acid is cultivated using methods well known to oneskilled in the art.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

Marker Assisted Selection

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants. To further speed up the backcrossingprogram, the embryo from immature seeds (25 days post anthesis) may beexcised and grown up on nutrient media under sterile conditions, ratherthan allowing full seed maturity. This process, termed “embryo rescue”,used in combination with DNA extraction at the three leaf stage andanalysis of at least one genetic variation that alters ROS1a activityand that confers upon the plant increased aleurone thickness, allowsrapid selection of plants carrying the desired trait, which may benurtured to maturity in the greenhouse or field for subsequent furtherbackcrossing to the recurrent parent.

Any molecular biological technique known in the art can be used in themethods of the present invention. Such methods include, but are notlimited to, the use of nucleic acid amplification, nucleic acidsequencing, nucleic acid hybridization with suitably labeled probes,single-strand conformational analysis (SSCA), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavageanalysis (CCM), catalytic nucleic acid cleavage or a combination thereof(see, for example, Lemieux, 2000; Langridge et al., 2001). The inventionalso includes the use of molecular marker techniques to detectpolymorphisms linked to alleles of the (for example) ROS1a gene whichalters ROS1a activity and that confers upon the plant increased aleuronethickness. Such methods include the detection or analysis of restrictionfragment length polymorphisms (RFLP), RAPD, amplified fragment lengthpolymorphisms (AFLP) and microsatellite (simple sequence repeat, SSR)polymorphisms. The closely linked markers can be obtained readily bymethods well known in the art, such as Bulked Segregant Analysis, asreviewed by Langridge et al. (2001).

In an embodiment, a linked loci for marker assisted selection is atleast within 1 cM, or 0.5 cM, or 0.1 cM, or 0.01 cM from a gene encodinga polypeptide of the invention.

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (M. J. McPherson and S. GMoller (editors), BIOS Scientific Publishers Ltd, Oxford, (2000)). PCRcan be performed on cDNA obtained from reverse transcribing mRNAisolated from plant cells expressing a ROS1a gene or allele which uponthe plant increased aleurone thickness. However, it will generally beeasier if PCR is performed on genomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.,(supra) and Sambrook et al., (supra). Sequencing can be carried out byany suitable method, for example, dideoxy sequencing, chemicalsequencing or variations thereof. Direct sequencing has the advantage ofdetermining variation in any base pair of a particular sequence.

Tilling

Plants of the invention can be produced using the process known asTILLING (Targeting Induced Local Lesions IN Genomes). In a first step,introduced mutations such as novel single base pair changes are inducedin a population of plants by treating seeds (or pollen) with a chemicalmutagen, and then advancing plants to a generation where mutations willbe stably inherited. DNA is extracted, and seeds are stored from allmembers of the population to create a resource that can be accessedrepeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cel I, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique.

TILLING is further described in Slade and Knauf (2005), and Henikoff etal. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Grain Processing

Due to the thickened aleurone, rice grain of the invention, and flourand bran therefrom, has an improved nutritional content. Isolatedaleurone tissue should contain low levels of starch and pericarp, andrepresents a major portion of the grain's physiologically beneficialsubstances for human nutrition. For instance, grain of the inventionand/or flour produced therefrom comprises, when compared to acorresponding wild-type grain and/or flour produced therefrom, one ormore or all of the following, each on a weight basis,

i) a higher mineral content such as about at least 20% or at least about25% higher, preferably the mineral content is the content one or more orall of zinc (such as at least about 10% or at least about 15% higher),iron (such as at least about 10% or at least about 15% higher),potassium (such as at least about 20% or at least about 25% higher),magnesium (such as at least about 18% or at least about 22% higher),phosphorus (such as at least about 17% or at least about 21% higher) andsulphur (such as at least about 5% or at least about 8% higher),

ii) a higher antioxidant content such as at least about 25%, or at leastabout 35%, more total phenolic compounds, and/or at least about 60%, orat least about 70%, more hydrophilic antioxidants,

iii) a higher phytate content such as at least about 10% or at leastabout 15% higher,

iv) a higher content of one or more or all of vitamins B3, B6 and B9,

v) a higher dietary fibre content and/or insoluble fibre content (suchas at least about 150%, or at least about 180%, higher total fibre),

vi) a starch content which is between about 90% and about 100% by weightrelative to the starch content of the corresponding wild-type grain,

vii) a higher sucrose content,

viii) a higher monosaccharide content (for example arabinose, xylose,galactose, glucose content) such as at least about 1.5 or at least about2 fold higher,

ix) higher fat content such as at least about 20%, at least about 30% orat least about 50%, or about 50%, higher, and

x) similar nitrogen levels.

Each of these nutritional components of grain can be determined usingroutine techniques such as outlined in Examples 1 and 5.

In one embodiment, rice grain of the invention and/or flour producedtherefrom, comprises one or more or all of the following, each on aweight basis,

i) at least about 20%, at least about 30% or at least about 50%, orabout 50% more fat when compared to corresponding wild-type grain/flour,

ii) at least about 11 mg/g or at least about 12 mg/g total phytate,

iii) at least about 20% or at least about 25% more mineral content inflour obtained from the grain when compared to flour from acorresponding wild-type grain,

iv) at least about 14 mg/kg or at least about 15 mg/kg total zinc,

v) at least about 13 mg/kg or at least about 13.5 mg/kg total iron,

vi) at least about 150%, or at least about 180% more total fibre whencompared to corresponding wild-type grain/flour,

vii) a starch content which is between about 90% and about 100% byweight relative to the starch content of the corresponding wild-typegrain/flour,

viii) at least about 1.5 or at least about 2 fold higher monosaccharidecontent (for example arabinose, xylose, galactose, glucose content) whencompared to corresponding wild-type grain/flour, and

ix) at least about 25%, or at least about 35%, more total phenoliccompounds, and/or at least about 60%, or at least about 70%, morehydrophilic antioxidants, when compared to corresponding wild-typegrain/flour.

In an embodiment, the grain comprises an increased proportion of amylosein its total starch content compared to the corresponding wild-typegrain. Methods of producing such grain are described in, for example, WO2002/037955, WO 2003/094600, WO 2005/040381, WO 2005/001098, WO2011/011833 and WO 2012/103594.

In an embodiment, grain of the invention comprises an increasedproportion of oleic acid and/or a decreased proportion of palmitic acidin its total fatty acid content compared to the corresponding wild-typegrain. Methods of producing such grain are described in, for example, WO2008/006171 and WO 2013/159149.

Grain/seed of the invention, or other plant parts of the invention, canbe processed to produce a food ingredient, food or non-food productusing any technique known in the art.

As used herein, the term “other food or beverage ingredient” refers toany substance suitable for consumption by an animal, preferably anysubstance suitable for consumption by a human, when provided as part ofa food or beverage. Examples include, but are not limited to, grain fromother plant species, sugar, etc, but excluding water.

In one embodiment, the product is whole grain flour such as, forexample, an ultrafine-milled whole grain flour, or a flour made fromabout 100% of the grain. The whole grain flour includes a refined flourconstituent (refined flour or refined flour) and a coarse fraction (anultrafine-milled coarse fraction).

Refined flour may be flour which is prepared, for example, by grindingand bolting cleaned grain. The particle size of refined flour isdescribed as flour in which not less than 98% passes through a clothhaving openings not larger than those of woven wire cloth designated“212 micrometers (U.S. Wire 70)”. The coarse fraction includes at leastone of: bran and germ. For instance, the germ is an embryonic plantfound within the grain kernel. The germ includes lipids, fiber,vitamins, protein, minerals and phytonutrients, such as flavonoids. Thebran includes several cell layers and has a significant amount oflipids, fiber, vitamins, protein, minerals and phytonutrients, such asflavonoids. Further, the coarse fraction may include an aleurone layerwhich also includes lipids, fiber, vitamins, protein, minerals andphytonutrients, such as flavonoids. The aleurone layer, whiletechnically considered part of the endosperm, exhibits many of the samecharacteristics as the bran and therefore is typically removed with thebran and germ during the milling process. The aleurone layer containsproteins, vitamins and phytonutrients, such as ferulic acid.

Further, the coarse fraction may be blended with the refined flourconstituent. The coarse fraction may be mixed with the refined flourconstituent to form the whole grain flour, thus providing a whole grainflour with increased nutritional value, fiber content, and antioxidantcapacity as compared to refined flour. For example, the coarse fractionor whole grain flour may be used in various amounts to replace refinedor whole grain flour in baked goods, snack products, and food products.The whole grain flour of the present invention (i.e. -ultrafine-milledwhole grain flour) may also be marketed directly to consumers for use intheir homemade baked products. In an exemplary embodiment, a granulationprofile of the whole grain flour is such that 98% of particles by weightof the whole grain flour are less than 212 micrometers.

In further embodiments, enzymes found within the bran and germ of thewhole grain flour and/or coarse fraction are inactivated in order tostabilize the whole grain flour and/or coarse fraction. Stabilization isa process that uses steam, heat, radiation, or other treatments toinactivate the enzymes found in the bran and germ layer. Flour that hasbeen stabilized retains its cooking characteristics and has a longershelf life.

In additional embodiments, the whole grain flour, the coarse fraction,or the refined flour may be a component (ingredient) of a food productand may be used to product a food product. For example, the food productmay be a bagel, a biscuit, a bread, a bun, a croissant, a dumpling, anEnglish muffin, a muffin, a pita bread, a quickbread, arefrigerated/frozen dough product, dough, baked beans, a burrito, chili,a taco, a tamale, a tortilla, a pot pie, a ready to eat cereal, a readyto eat meal, stuffing, a microwaveable meal, a brownie, a cake, acheesecake, a coffee cake, a cookie, a dessert, a pastry, a sweet roll,a candy bar, a pie crust, pie filling, baby food, a baking mix, abatter, a breading, a gravy mix, a meat extender, a meat substitute, aseasoning mix, a soup mix, a gravy, a roux, a salad dressing, a soup,sour cream, a noodle, a pasta, ramen noodles, chow mein noodles, lo meinnoodles, an ice cream inclusion, an ice cream bar, an ice cream cone, anice cream sandwich, a cracker, a crouton, a doughnut, an egg roll, anextruded snack, a fruit and grain bar, a microwaveable snack product, anutritional bar, a pancake, a par-baked bakery product, a pretzel, apudding, a granola-based product, a snack chip, a snack food, a snackmix, a waffle, a pizza crust, animal food or pet food.

In alternative embodiments, the whole grain flour, refined flour, orcoarse fraction may be a component of a nutritional supplement. Forinstance, the nutritional supplement may be a product that is added tothe diet containing one or more additional ingredients, typicallyincluding: vitamins, minerals, herbs, amino acids, enzymes,antioxidants, herbs, spices, probiotics, extracts, prebiotics and fiber.The whole grain flour, refined flour or coarse fraction of the presentinvention includes vitamins, minerals, amino acids, enzymes, and fiber.For instance, the coarse fraction contains a concentrated amount ofdietary fiber as well as other essential nutrients, such as B-vitamins,selenium, chromium, manganese, magnesium, and antioxidants, which areessential for a healthy diet. For example 22 grams of the coarsefraction of the present invention delivers 33% of an individual's dailyrecommend consumption of fiber. The nutritional supplement may includeany known nutritional ingredients that will aid in the overall health ofan individual, examples include but are not limited to vitamins,minerals, other fiber components, fatty acids, antioxidants, aminoacids, peptides, proteins, lutein, ribose, omega-3 fatty acids, and/orother nutritional ingredients. The supplement may be delivered in, butis not limited to the following forms: instant beverage mixes,ready-to-drink beverages, nutritional bars, wafers, cookies, crackers,gel shots, capsules, chews, chewable tablets, and pills. One embodimentdelivers the fiber supplement in the form of a flavored shake or malttype beverage, this embodiment may be particularly attractive as a fibersupplement for children.

In an additional embodiment, a milling process may be used to make amulti-grain flour or a multi-grain coarse fraction. For example, branand germ from one type of grain may be ground and blended with groundendosperm or whole grain cereal flour of another type of cereal.Alternatively bran and germ of one type of grain may be ground andblended with ground endosperm or whole grain flour of another type ofgrain. It is contemplated that the present invention encompasses mixingany combination of one or more of bran, germ, endosperm, and whole grainflour of one or more grains. This multi-grain approach may be used tomake custom flour and capitalize on the qualities and nutritionalcontents of multiple types of cereal grains to make one flour.

It is contemplated that the whole grain flour, coarse fraction and/orgrain products of the present invention may be produced by any millingprocess known in the art. An exemplary embodiment involves grindinggrain in a single stream without separating endosperm, bran, and germ ofthe grain into separate streams. Clean and tempered grain is conveyed toa first passage grinder, such as a hammermill, roller mill, pin mill,impact mill, disc mill, air attrition mill, gap mill, or the like. Aftergrinding, the grain is discharged and conveyed to a sifter. Further, itis contemplated that the whole grain flour, coarse fraction and/or grainproducts of the present invention may be modified or enhanced by way ofnumerous other processes such as: fermentation, instantizing, extrusion,encapsulation, toasting, roasting, or the like.

EXAMPLES Example 1. General Materials and Methods

Observation of Aleurone by Staining with Sudan Red Solution

Stain solution was prepared by adding 1 g of Sudan red IV to 50 ml ofpolyethylene glycol solution (average molecular weight 400, Sigma, Cat.No. 202398), incubated at 90° C. for one hour, and mixed with equalvolume of 90% glycerol. After removing the fruit coat (palea and lemma)of each grain, mature rice grains were incubated in distilled water forfive hours and then sectioned transversely or longitudinally using arazor blade. Sections were stained in Sudan red solution at roomtemperature for 24 to 72 hours. The sections were then counter-stainedwith Lugol staining solution at room temperature for 20 min (Sigma,32922) and observed under dissecting microscope (Sreenivasulu, 2010).

Staining of Aleurone with Evans Blue

Evans Blue stain solution was prepared by dissolving 0.1 g of Evans blue(Sigma, E2129) in 100 ml distilled water. After removing the fruit coat(palea and lemma) of each grain, mature rice grains were sectionedtransversely using a razor blade. Sections were incubated in distilledwater at room temperature for 30 min, the stain added and left at roomtemperature for 2 min. The stain solution was then discarded, thesections washed twice with distilled water and observed under adissecting microscope.

Light Microscopic Observation of Rice Endosperm

Rice grains were fixed in formalin-acetic acid-alcohol (FAA) solution(60% ethanol, 5% glacial acetic acid and 2% formaldehyde), degassed forone hour, dehydrated in a series of alcohol solutions containing 70%,80%, 95% and then 100% ethanol, infiltrated by LR white resin (ElectronMicroscopy Sciences, 14380) and polymerized for 24 hours at 60° C.Microtome sectioning was done using a Leica UC7 microtome. Sections werestained in 0.1% toluidine blue solution (Sigma, T3260) at roomtemperature for 2 min, then washed twice with distilled water andexamined by light microscopy. Alternatively, sections were stained in0.01% Calcofluor white solution (Sigma, 18909) at room temperature for 2min and examined by light microscopy.

Staining with PAS and Commassie Blue

The fixed sections on slides were incubated in preheated 0.4% periodicacid (Sigma, 375810) at 57° C. for 30 min, then rinsed three times indistilled water. Schiff reagent (Sigma, 3952016) was applied and theslides incubated at room temperature for 15 min, then rinsed three timesin distilled water. The sections were then incubated in 1% Coomassieblue (R-250), ThermoScientific, 20278) at room temperature for 2 min,and rinsed three times in distilled water. Dehydration of the sectionswas achieved using a series of alcohol solutions having 30%, 50%, 60%,75%, 85%, 95% to 100% ethanol for 2 min each, followed by clearing ofeach slide in 50% xylene and 100% xylene solution (Sigma, 534056) for 2min each. Coverslips were then mounted with Eukitt® quick hardeningmounting medium (Fluka, 03989) and the sections observed under a lightmicroscope.

DNA Extraction and PCR Conditions

Two methods were used for DNA extraction from plant leaf samples—a rapidDNA extraction method to provide less pure DNA samples and a moreextensive DNA extraction method for purer DNA, modified from Huang(2009). In the first method, four glass beads with diameter of 2 mm(Sigma, 273627), 1 to 2 mg of rice leaf tissue and 150 μl of extractionbuffer (10 mM Tris, pH9.5, 0.5 mM EDTA, 100 mM KCl) were added to eachwell of a 96-well PCR plate. The plate was sealed and mixtureshomogenised using a Mini-Beadbeater-96 mixer (GlenMills, 1001) for 1min. After centrifugation at 3000 rpm for 5 min, the extractedsupernatants containing DNA were used in PCR reactions.

In the second method, two glass beads with diameter of 2 mm and 0.2 gleaf were in 1.5 ml Eppendorf tubes were cooled in liquid nitrogen for10 min. Samples were then homogenised in the Mini-Beadbeater-96 for 1min, then 600 μl DNA extraction buffer (2% SDS, 0.4M NaCl, 2 mM EDTA, 10mM Tris-HCl, pH8.0) was added to each tube and the mixtures incubated at65° C. for one hour. After cooling the mixtures, 450 d of 6M NaCl wasadded, mixed and centrifuged at 12000 rpm for 20 min. Each supernatantwas transferred to a new tube and the DNA precipitated using an equalvolume of 2-propanol at −20° C. for one hour. DNA was recovered bycentrifugation at 2400 rpm at 4° C. for 20 min and the pellets washedtwice with 75% ethanol. The pellets were air-dried at room temperatureand each resuspended in 600 μl distilled water containing 10 ng/ulRNAse(ThermoScientific, EN0201)) and used in PCR reactions.

The PCR reactions used 5 μl of 2×PCR buffer containing Taq Polymerase(ThermoScientific, K0171), 5′ and 3′ oligonucleotide primers and 1 μl ofDNA sample in a total volume of 10 μl. Amplification was performed using35 cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 30 sec.Amplification products were analysed by gel electrophoresis using 3%agarose gels. Control PCR reactions used DNA preparations fromhomozygous Zhonghua11 (ZH11) (wild-type japonica rice), homozygous NJ6(wild type indica rice), and the mixture of ZH11 and NJ6.

For genetic mapping of the ta2 allele, PCR amplifications for thegenetic markers used the following primer pairs (5′ to 3′ sequences):INDEL 127 (position 6,343,260 on Chromosome 1), forward primerTGAGTAGTTGCGTTGTTCT (SEQ ID NO: 15), reverse primer TCTTAGTGAGCCGTTTCT(SEQ ID NO: 16); INDEL 129 (position 6,560,681 on Chromosome 1), forwardprimer CCTTCTGTGCTATGGGTT (SEQ ID NO: 17), reverse primerCATGCCAAGACACCACTT (SEQ ID NO: 18); INDEL 128 (position 6,470,027 onChromosome 1), forward primer TGGCTTTGGAAACGGTAG (SEQ ID NO: 19),reverse primer TTTAGAGGGATGTGCGTCA (SEQ ID NO: 20); INDEL 149 (position6,427,144 on Chromosome 1), forward primer AAACAACGATCCAGCAAA (SEQ IDNO: 21), reverse primer TTGGCACCGTATTACTTTC (SEQ ID NO: 22).

TILLING Assays

The primers that were used in the TILLING assays had the nucleotidesequences:

TA2-1F: (SEQ ID NO: 23) ACGCATTCTTCATTGACTGTATGT TA2-1R: (SEQ ID NO: 24)GCCCTTTCAATACAATGACTAGGT TA2-2F: (SEQ ID NO: 25)GAACATTTGAATCATGTTCCTCAC TA2-2R: (SEQ ID NO: 26)ACTATCCTTTGATGCAAGTTCTCC TA2-3F: (SEQ ID NO: 27)GTTGGAAGAGCAGTTAAAGCAAAT TA2-3R: (SEQ ID NO: 28)CTTCGGCAGTGAAATTTAGTAACA TA2-4F: (SEQ ID NO: 29)TACAGAACTTCTACGAATGCAGGA TA2-4R: (SEQ ID NO: 30)GCAACATGAATTGCTAAAGATGAG.

The PCR amplifications with ExTaq were performed with the followingreaction conditions: 95° C. for 2 min; 8 cycles of 94° C. for 20 s, 68°C. for 30 s (1° C. decrease per cycle), and 72° C. for 60 s for every 1kb of amplicon length, followed by 35 cycles of 94° C. for 20 s, 60° C.for 30 s, and 72° C. for 60 s for each 1 kb of amplicon length, and afinal extension at 72° C. for 5 min. PCR products from the wild-type andtest samples were mixed and subjected to a complete denaturation-slowannealing program to form heteroduplexes under the following conditions:99° C. for 10 min for denaturation, followed by 70 cycles of decrements,starting at 70° C., 20 s each, with a 0.3° C. decrease per cycle, andthen holding at 15° C. to reanneal the denatured PCR products to formheteroduplexes. Cell digestions of annealed PCR products were performedin 15 μL reaction mixtures containing Cell buffer (10 mM HEPES, pH 7.5,10 mM KCl, 10 mM MgSO₄, 0.002% Triton X-100, and 0.2 μg/mL bovine serumalbumin (BSA), 4 μL of PCR product, and 1 unit Cell (10 units/μL) if PCRproducts were polymerized by Ex Taq, or 20 units Cell if the PCRproducts were polymerized by KOD), at 45° C. for 15 min, followed byadding 3 μL of 0.5 M EDTA (pH 8.0) to stop the reaction. Alternatively,the digestions were performed in 15-μL reaction mixtures containing 4 μLof PCR products and 2 units of mung bean nuclease (MBN, 10 units/μL,Cat. No. M0250S; New England Biolabs, USA) in MBN buffer (20 mMBis-Tris, pH 6.5, 10 mM MgSO₄, 0.2 mM ZnSO₄, 0.002% Triton X-100, and0.2 μg/mL BSA) at 60° C. for 30 min, followed by adding 2 μL of 0.2% SDSto stop the reaction.

Cell-digested PCR products in 96-well PCR plates were diluted to 100 μLwith deionized water, and capillary electrophoresis was performed at 9kV, 30 s for pre-run, 15 s for injection of 1 ng/μL molecular weightmarker 75 and 15 kb or 50 and 3 kb dsDNA (Fermentas, Canada), 45 s forsample injection, and 40 min for sample separations in an AdvanCE™ FS96apparatus (Advanced Analytical Technologies, USA). Gel pictures wereacquired and analysed using PROSize software (Advanced AnalyticalTechnologies, USA) for capillary electrophoresis.

DNA Glycosylase (DME) Enzyme Assays

Demeter (DME) is a bifunctional DNA glycosylase/lyase with activity on5-methylcytosine substrates. Plants have 5-methylcytosine in the threesequence contexts: CpG, CpNpG, and CpNpN and DME has activity on5-methylcytosine in each of these sequence contexts. In the enzyme assaywhich is performed in vitro, the cleavage of the phosphodiester linkageon the 5′ side of a methylated cytosine was detected, yielding δelimination products. Treatment of the DNA reaction products with strongbase (NaOH) prior to gel electrophoresis confirmed the δ eliminationprocess at the predicted position.

Synthetic oligonucleotides which were to be used as substrates in theenzyme assays were synthesized as follows with nucleotide modificationsdenoted within parentheses as shown below:

MEA-1.6F, (SEQ ID NO: 31) 5′-CTATACCTCCTCAACTCCGGTCACCGTCTCCGGCGMEA-1.6F18meC, (SEQ ID NO: 32)5′-CTATACCTCCTCAACTC(5-meC)GGTCACCGTCTCCGGCG MEA-1.6F17meC, (SEQ ID NO:33) 5′-CTATACCTCCTCAACT(5-meC)CGGTCACCGTCTCCGGCG MEA-1.6F22meC, (SEQ IDNO: 34) 5′-CTATACCTCCTCAACTCCGGT(5-meC)ACCGTCTCCGGCG MEA-1.6F18AP, (SEQID NO: 35) 5′-CTATACCTCCTCAACTC(abasic)GGTCACCGTCTCCGGCG MEA-1.6F17AP,(SEQ ID NO: 36) 5′-CTATACCTCCTCAACT(abasic)CGGTCACCGTCTCCGGCGMEA-1.6F15AP, (SEQ ID NO: 37)5′-CTATACCTCCTCAA(abasic)TCCGGTCACCGTCTCCGGCG MEA-1;6F12AP, (SEQ ID NO:38) 5′-CTATACCTCCT(abasic)AACTCCGGTCACCGTCTCCGGCG MEA-1.6F18T, (SEQ IDNO: 39) 5′-CTATACCTCCTCAACTCTGGTCACCGTCTCCGGCG MEA-1.6R, (SEQ ID NO: 40)5′-CGCCGGAGACGGTGACCGGAGTTGAGGAGGTATAG MEA-1.6R17meC, (SEQ ID NO: 41)5′-CGCCGGAGACGGTGAC(5-meC)GGAGTTGAGGAGGTATAG.

Twenty pmol of each oligonucleotide was end-labelled in a 50 μL reactionusing 20 units of T4 polynucleotide kinase in the presence of 30 μCi of(γ-32P)-ATP (6000 Ci/mmol, Perkin Elmer Life Sciences) at 37° C. for 1hr. Each labelled oligonucleotide was purified using a QiaquickNucleotide Removal Kit (Qiagen) as described by the manufacturer.Labelled oligonucleotides were annealed to the appropriate complementaryoligonucleotides in 10 mMTris-HCl (pH 8.0), 1 mM EDTA and 0.1 M NaCl.Each mixture was heated to 100° C. for 10 min and then slowly cooled toroom temperature overnight. MspI or HpaII restriction endonucleasedigestion followed by gel electrophoresis was used to determine theefficiency of annealing. Only substrates that were greater than 90%double-stranded were used in glycosylase activity assays.

5′-labeled oligonucleotide substrates (13.3 nM) were incubated with DMEprotein (250 nM) in a 15 ml reaction with 40 mM HEPES-KOH (pH 8.0), 0.1MKCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 200 mg/ml BSA at 370 for 1hr. The reaction was terminated with 15 ml of 95% formamide, 20 mM EDTA,0.05% bromophenol blue, 0.05% xylene cyanol FF and boiled for 5 min. Toinduce elimination, NaOH was added at a final concentration of 0.1 M andthe reaction was boiled for 7 min. Products were fractionated on a 15%polyacrylamide gel containing 7.5 M urea and 1×TBE. Electrophoresis wasdone at 1000V for 4 hr with a Hoefer SQ3 gel apparatus. The gel wasexposed to Kodak BioMax MR film at −80° C.

Analytical Methods

Proximates and other major constituents in grain, food ingredients andfood samples were determined using standard methods, for example asdescribed below.

Grain moisture content was measured according to (Association ofOfficial Analytical Chemists) AOAC Method 925.10. Briefly, grain samples(˜2 g) were dried to constant weight in an oven at 130° C. for about 1h.

Ash content was measured according to AOAC Method 923.03. Samples usedfor moisture determination were ashed in a muffle furnace at 520° C. for15 h.

Protein Content of Grain, Food Ingredients and Food Samples

Protein content was measured according to AOAC Method 992.23. Briefly,total nitrogen was analysed by the Dumas combustion method using anautomated nitrogen analyser (Elementar Rapid N cube, ElementarAnalysensysteme GmbH, Hanau, Germany). The protein content of grain orfood samples (g/100 g) was estimated by multiplying nitrogen content by6.25.

Sugars, Starch and Other Polysaccharides

Total starch content was measured according to AOAC Method 996.11 whichuses the enzymatic method of McCleary et al. (1997).

The amount of sugars was measured according to AOAC Method 982.14.Briefly, simple sugars were extracted with aqueous ethanol (80% ethanol)and then quantified by HPLC using a polyamine-bonded polymeric gelcolumn, using acetonitrile:water (75:25 v/v) as the mobile phase and anevaporative light scattering detector.

Total neutral non-starch polysaccharides (NNSP) were measured by the gaschromatographic procedure of Theander et al. (1995) with a slightmodification which involved a 2 h hydrolysis with 1 M sulfuric acidfollowed by centrifugation.

Fructans (fructo-oligosaccharides) were analysed by the method detailedby AOAC Method 999.03. Briefly, the fructo-oligosaccharides wereextracted into water followed by digestion with asucrase/maltase/invertase mixture. The resultant free sugars were thenreduced with sodium borohydride and digested to fructose/glucose withfructanose. The released fructose/glucose was measured usingp-hydroxybenzoic acid hydrazine (PAHBAH).

Fibre Content

Total Dietary Fibre (TDF) was measured according to AOAC Method 985.29and Soluble and Insoluble Fibre (SIF) according to AOAC Method 991.43.Briefly, TDF was determined by the gravimetric technique of Prosky etal. (1985), as detailed in the AOAC Method 985.29, and SIF wasdetermined by a gravimetric technique as described in AOAC 991.43.

Total Lipid

Samples of 5 g flour were incubated with 1% Clarase 40000 (SouthernBiological, MC23.31) at 45° C. for one hour. Lipids were extracted fromthe samples into chloroform/methanol by multiple extractions. Aftercentrifugation to separate phases, the chloroform/methanol fraction wasremoved and dried at 101° C. for 30 min to recover the lipid. The massof residue left represents the total lipid in the sample (AOAC Method983.23).

Fatty Acid Profile of Lipids

Lipid was extracted from milled flours into chloroform according to AOACMethod 983.23. A portion of the chloroform fraction containing the lipidwas evaporated under a stream of nitrogen after addition of an aliquotof hepta-decanoic acid as an internal standard. The residue wassuspended in 1% sulfuric acid in dry methanol and the mixture heated at50° C. for 16 h. The mixture was diluted with water and extracted twicewith hexane. The combined hexane solution was loaded onto a small columnof Florisil and the column washed with hexane and the fatty acid methylesters then eluted with 10% ether in hexane. The eluent was evaporatedto dryness and the residue dissolved in iso-octane for injection ontothe GC. Fatty acid methyl esters were quantified against a mixture ofstandard fatty acids. GC conditions: Column SGE BPX70 30 m×0.32 mm×0.25um; Injection 0.5 μL; Injector 250° C.; 15:1 split; Flow 1.723 ml/minconstant flow; Oven 150° C. for 0.5 min, 10° C./min to 180° C., 1.5°C./min to 220° C., 30° C./min to 260° C. (total run-time 33 mins);Detector FID at 280° C.

Antioxidant Activity (ORAC-H)

The hydrophilic antioxidant activity (ORAC-H) was determined followingthe method of Huang (2002a and 2002b) with modification as described byWolbang et al. (2010). The samples were extracted for lipophilicantioxidants followed by hydrophilic antioxidants as follows: 100 mg ofsample weighed in triplicate into 2 mL microtubes. 1 mLhexane:dichloromethane (50:50) was then added and mixed vigorously for 2min and centrifuged at 13,000 rpm for 2 min at 10° C. The supernatantwas transferred to a glass vial and the pellet re-extracted with afurther 2 mL of hexane:dichloromethane mix. The mixing and centrifugesteps were then repeated, and the supernatant transferred to the sameglass vial. Residual solvent from the pellet was evaporated under agentle stream of nitrogen. 1 mL of acetone:water:acetic acid mix(70:29.5:0.5) then added and mixed vigorously for 2 min. The mixture wasthen centrifuged as before and the supernatant used in the ORAC-H plateassay. Samples were diluted as required with phosphate buffer. The areaunder the curve (AUC) was calculated and compared against AUC values forTrolox standards. The ORAC value is reported as uMTrolox equivalents/gof sample.

Phenolics

Total phenolics content as well as phenolics in the free, conjugated andbound states were determined following extraction according to themethod described by Li et al. (2008) with minor modifications. Briefly,the free phenolics were determined in 100 mg samples followingextraction into 2 mL 80% methanol by sonication for 10 mins in a glassvial (8 ml capacity). The supernatant was transferred to a second glassvial and the extraction of the residue repeated. The combinedsupernatants were evaporated to dryness under nitrogen. 2 mL of aceticacid (2%) was added to adjust the pH to about 2 and then 3 mL ethylacetate added to extract the phenolics with shaking for 2 mins. Thevials were centrifuged at 2000×g for 5 mins at 10° C. Supernatants weretransferred to a clean glass vial and the extraction repeated twicemore. Combined supernatants were evaporated under nitrogen at 37° C.Residues were dissolved in 2 mL 80% methanol and refrigerated.

Samples for the conjugated phenolics were treated as for the freephenolic assay for the initial 80% methanol extraction. At this point2.5 mL (2M) sodium hydroxide and a magnetic bar were added to theevaporated supernatants in the glass vial which was then filled withnitrogen and capped tightly. The vials were mixed and heated at 110° C.for 1 h with stirring. Samples were cooled on ice before extraction with3 mL ethyl acetate by shaking for 2 min. The vials were centrifuged at2000×g for 5 min at 10° C. Supernatants were discarded and pH adjustedto about 2 with 12 M HCL. Phenolics were extracted using 3×3 mL aliquotsof ethyl acetate as described for the free phenolics. The supernatantswere combined and evaporated to dryness under nitrogen at 37° C. and theresidue was taken up in 2 mL 80% methanol and refrigerated.

Bound phenolics were measured from the residues following methanolicextraction of the free phenolics. 2.5 mL (2M) sodium hydroxide and amagnetic bar were added to the residue before filling the vial withnitrogen and capping it tightly. The vials were mixed and heated at 110°C. for 1 h with stirring. Samples were cooled on ice before extractionwith 3 mL ethyl acetate by shaking for 2 mins. The vials werecentrifuged at 2000×g for 5 mins at 10° C. The supernatant was discardedand pH adjusted to about 2 with 12 M HCl. Phenolics were extracted with3×3 mL aliquots of ethyl acetate as described for the free phenolics.The supernatants were combined and evaporated to dryness under nitrogenat 37° C. and the residue was taken up in 2 mL 80% methanol andrefrigerated.

Total phenolics were determined using 100 mg of samples by adding 200 uL80% methanol to wet the samples prior to hydrolysis. 2.5 mL (2M) sodiumhydroxide and a magnetic bar were added before filling the vial withnitrogen and capping tightly. The vials were mixed and heated at 110° C.for 1 hr with stirring. Samples were cooled on ice before extractionwith 3 mL ethyl acetate by shaking for 2 mins. The vials werecentrifuged at 2000×g for 5 mins at 10° C. The supernatant was discardedand pH adjusted to about 2 with 12 M HCl. Phenolics were extracted with3×3 mL aliquots of ethyl acetate as described for the free phenolics.The supernatants were combined and evaporated to dryness under nitrogenat 37° C. and the residue was taken up in 4 mL 80% methanol andrefrigerated.

The amount of phenolics in the treated/extracted samples was measuredusing Folin Ciocalteu's assay for determination of phenolics. Gallicacid standards at 0, 1.56, 3.13, 6.25, 12.5, and 25 μg/mL were used toprepare a standard curve. 1 mL of standards were added to 4 mL glasstubes. For test samples, 100 μL aliquots of thoroughly mixed sampleswere added to 900 μL water in 4 mL glass tubes. 100 mL of FolinCiocalteu's reagent was then added to each tube which was vortexedimmediately. 700 μL sodium bicarbonate solution (1 M) was added after 2min and then mixed by vortexing. Each solution was incubated at roomtemperature in the dark for 1 h and then absorbance read at 765 nm.Results were expressed in μg gallic acid equivalents/g sample.

Phytate

Determination of the phytate content of the flour samples was based onthe method of Harland and Oberleas, as described in AOAC OfficialMethods of Analysis (1990). Briefly, a 0.5 g flour sample was weighedand extracted with 2.4% HCl using a rotating wheel (30 rpm) for 1 hourat room temperature. The mixture was then centrifuged at 2000×g for 10minutes and the supernatant extracted and diluted 20-fold with milli-Qwater. An anion exchange column (500 mg Agilent Technologies) was placedon a vacuum manifold and conditioned for use following themanufacturer's instructions. The diluted supernatant was then loadedonto a column and non-phytate species removed by washing with 0.05 MHCl. Phytate was then eluted with 2 M HCl. The collected eluate wasdigested using a heating block. The sample was cooled and the volumemade up to 10 mL with milli-Q water. Phosphorous levels were determinedby spectrophotometer using the molybdate, sulphonic acid colouringmethod with absorbance readings at 640 nm. Phytate was calculated usingthe following formula:Phytate (mg/g)=P conc*V1*V2/(1000*sample weight*0.282)where P cone is the concentration of phosphorous (μg/mL), as determinedby spectrophotometry, V1 is the volume of the final solution, V2 is thevolume of the extracted phytate solution, and 0.282 is the phosphorus tophytate conversion factor.Total Mineral Content Estimation

Total mineral content of samples was measured by ash assay using AOACMethods 923.03 and 930.22. About 2 g of flour was heated at 540° C. for15 hours and the mass of ash residue was then weighed. Wholemeal floursamples of 0.5 g were digested using tube block digestion with 8M nitricacid at 140° C. for eight hours. Zinc, iron, potassium, magnesium,phosphorus and sulphur contents were then analysed using inductivelycoupled plasma atomic emission spectrometry (ICP-AES) according toZarcinas (1983a and 1983b).

Minerals were analysed at CSIRO, Urrbrae, Adelaide South Australia, atWaite Analytical Service (University of Adelaide, Waite, SouthAustralia) and at Dairy Technical Services (DTS, North Melbourne,Victoria). Elements were determined by Inductively CoupledPlasma-Optical Emission Spectroscopy (ICP-OES) after digestion withnitric acid solution (CSIRO) or dilute nitric acid and hydrogen peroxide(DTS) or by Inductively Coupled Plasma Atomic Emission Spectroscopy(ICP-AES) after digestion with nitric/perchloric acid solution.

Vitamins

Vitamins B1 (Niacin), B3 (Pyridoxine) and total folate analyses wereperformed by DTS as well as National Measurement Institute (NMI). Niacinwas measured by AOAC Methods 13th Ed (1980) 43.045, according to Lahey,et al. (1999). Pyridoxine was measured according to Mann et al. (2001).The method incorporated a pre-column transformation of phosphorylatedand free vitamin B6 forms into pyridoxine (pyridoxol). Acid phosphatasehydrolysis was used for dephosphorylation followed by de-amination withglyoxylic acid in the presence of Fe²⁺ to convert pyridoxamine topyridoxal. Pyridoxal was then reduced by sodium borohydride topyridoxine.

Folic acid was measured either according to VitaFast Folic acid kitusing the manufacturer's instructions, or according to AOAC method2004.05.

Example 2. Isolation and Characterisation of Thick-Aleurone (ta) Mutants

Establishment and Cultivation of a Mutagenised Rice Population

About 8000 grains (designated M₀ grains) from wild-type rice cultivarZhonghua11 (ZH11) were mutagenized by treatment with 60 mM ethyl methanesulfonate (EMS) using standard conditions. Mutagenised grains were sownin the field and the resultant plants cultivated to produce M₁ grains.M₁ grains were harvested and then sown in the field to produce M₁plants. 8925 panicles were harvested from 1327 individual M₁ plants.From these plants 36,420 M₂ grains were screened, including at east 4grains from each panicle.

Mutant Screening by Staining of Half Grains

The fruit coat (palea and lemma) of M₂ rice grains were removed. Each ofthe 36,420 grains was transverse bisected. The halves containing anembryo were saved in 96-well plates for subsequent germination, whileeach half grain without an embryo was stained with Evans Blue andobserved under a dissecting microscope to detect mutant grains havingthickened aleurones relative to the wild-type. The staining was based onthe principle that Evans Blue could only penetrate and stain non-viablecells such as the cells of the starchy endosperm while no colour changewas observed in the viable aleurone layer. From initial Evans Bluestaining and histological analyses, individual grains exhibitingsignificant increases in aleurone thickness as well as grains showing asignificant thickening in the ventral side aleurone of the seed wereobserved. Other grains showed an increase in aleurone thickness but to alesser extent. The unstained region of the ventral side of each seed wasespecially examined for thickness of the aleurone layer. Variants withincreases in thickness of the aleurone layer on the dorsal side of thegrains were also observed. Only variants with significant increases inthe thickness of the aleurone layer across the entire cross-section werechosen for further analysis.

Compared with wild-type half-grains, the half-grains having a thickerunstained region with Evans Blue were selected. Amongst the 36,420grains examined, 219 grains (0.60%) having differences in aleuronethickness were identified and selected. These had been obtained from 162panicles from 140 individual M₁ plants, and therefore most representedindependent mutants. One mutant grain in particular was identified andcharacterised further as described below, having a mutation designatedthick-aleurone 2 (ta2). The corresponding wild-type gene was thereforedesignated Ta2; that designation is used herein.

To maintain the putative mutant lines, each corresponding embryonatedhalf grain was germinated on medium containing half-strength MS saltsmedium (Murashige and Skoog, 1962) solidified with 1% Bacto agar (Bacto,214030) and cultured at 25° C. under light of intensity 1500˜2000 Luxwith 16 h light/8 h dark cycles. The plantlets were transferred to soilat the two to three leaflet stage and the resultant plants grown tomaturity. Upon the germination and cultivation of the correspondingembryonated half grains, 115 seeds (52.5% survival) were grown up toproduce mature and fertile plants.

Candidate mutant plants which exhibited little or no defects in generalagronomical traits such as those that were of normal plant height,fertility (male and female fertility), grain size and 1000 grain weightrelative to the wild-type parental variety as well as showing stableinheritance of the thickened aleurone trait were identified, selectedand further analysed. Among them, a mutant designated ta2 which showed amore extreme multi-aleurone phenotype of six to seven cell layers wasselected and analysed in detail. The wild-type grains exhibited analeurone of one cell layer, as expected.

Histological Analyses of the Ta2 Mutant Grains

Developing grain from wild-type ZH11 and ta2 mutant plants were studiedand compared for morphological changes from 1 to 30 days afterpollination (DAP). The ripening phase of rice grain can be said to havethree stages: a milk grain stage, a dough grain stage and a mature grainstage. In the dough grain stage, the grains in wild-type panicles beganto change in colour from green to yellow, following by a gradualdestruction of vesicular tissue connecting the stalk and caryopsis.Grains in the ta2 panicles were delayed in the colour change.Microscopic examination of the transverse sections of the rice grainsalso showed an increase in the degree of chalkiness (opaqueness) in ta2mutant grains. Scanning electron microscopy (SEM) was then used to studythe structure of the starch granule organization in the middle part ofthe starchy endosperm. In wild-type grains, starch granules were tightlypacked and showed a smooth surface and regular shape, while in ta2grains, a looser packing of irregular-shaped starch granules wasobserved. In summary, at least three changes were observed in the plantsand grain having the ta2 mutation: a delay in grain maturation, anincrease in the degree of chalkiness of the grain and in starch granulestructure.

Developing mutant and wild-type grains at 6, 7, 8, 9, 10, 12, 15, 18,21, 24, 27 and 30 DAP were stained with Evans Blue and the aleuronelayers examined by light microscopy. No significant difference wasobserved in the thickness of the developing aleurone layers betweenwild-type and ta2 mutant grains up until 10 DAP. After 10 DAP, thealeurone layers of ta2 mutants were thicker than in the wild-typegrains, and the difference reached a maximum at around 20 DAP. Theseresults were consistent with those from Sudan red staining.

The wild-type and ta2 mutant grains (30 DAP) were further examined forhistological differences by sectioning (1 μm), staining and lightmicroscopy. After staining with 0.1% toluidine blue which stains nucleicacid blue and polysaccharide purple, a single layer of large, regularlyoriented, rectangular cells was observed in wild-type aleurones. Incontrast, sections of ta2 mutant grains had aleurone layers of six toeight cell layers, the cells also being of varying sizes and irregularorientation. These observations indicated that the thickened aleuronesin the ta2 grains were mainly caused by the increase in the number ofcell layers rather than the enlargement of individual aleurone cells.

Further staining with 0.01% Calcoflour White, a fluorescent cell wallstain, showed no difference in cell wall thickness between wild-type andta2 mutant grains. The cell walls of aleurone cells were thicker thancell walls in the starchy endosperm for both wild-type and ta2 grains.

Analysis of the Agronomical Characteristics of Ta2 Mutant Plants andGrains

After backcrossed to wild-type (ZH11) plants for three generations inthe field to yield the BC3F3 generation, thereby removing additional,unlinked mutations that might have arisen from the mutagenic treatment,ta2 mutant plants were analysed for some agronomical traits. The ta2mutant plants and grain were not significantly different, compared towild-type plants and grain, in plant height, 1000 grain-weight, grainsize (length, width and thickness) and caryopsis morphology (Table 2).In contrast, wild-type plants showed a seed setting rate of 98.9%whereas ta2 mutant plants showed a decrease in seed setting rate at73.4%. The seed setting rate was calculated as the percentage of floretsin the plant that were filled by a seed by the mature grain stage.Moreover, the ta2 mutant grains showed a decrease in germinationcapacity of 75.1% in comparison with wild-type grain of 97.3% whencultured at 28° C. under 12 h light/12 h dark cycles without humiditycontrol in a growth chamber. Germination was defined as when the radiclehad visibly emerged through the seed coat.

Example 3. Genetic Analysis of the Ta2 Mutant

Based on the maternal origin of aleurone and endosperm tissues in agrowing plant, two genetic experiments were performed to determinewhether the thick aleurone phenotype was maternally determined. Firstly,a test cross was performed between a maternal ta2 (mutant) plant and apaternal wild-type plant, and F2 progeny grains obtained. Of the F2grains, 49.4% (n=634) showed the thickened aleurone phenotype, whichdeviated significantly from the 3:1 (wild-type:mutant) ratio predictedfor Mendelian inheritance of a dominant gene in an F2 population.Secondly, a reciprocal cross was performed between a ta2 plant and awild-type plant. All F1 seeds (100%; n=589) showed the thickenedaleurone phenotype, while in the reciprocal cross using the wild-type asthe maternal plant, all F1 seeds (n=197) resulted in the wild-typephenotype. These crosses showed that the aleurone phenotype wasdetermined by the maternal plant genotype.

TABLE 2 Comparison of wild-type (ZH11) and ta2 mutant plants foragronomical traits. ZH11 ta2 Plant Height (cm) 103.7 (±3.24) 112.2(±6.05) Seed setting rate (%) 98.9 (±3.41) 73.41 (±3.41) 1000 seedsweight (g) 22.73 (±0.17) 22.07 (±0.33) Seed length (mm) 7.46 (±0.26)7.55 (±0.27) Seed width (mm) 3.27 (±0.12) 3.15 (±0.10) Seed thickness(mm) 2.35 (±0.10) 2.27 (±0.11)

In order to establish whether the maternal effect was determined by thegametophytic or sporophytic genotype, F1 plants which were heterozygousTa2/ta2, obtained from a cross between homozygous Ta2/Ta2 and homozygousta2/ta2 plants, were used in reciprocal crosses with either ta2homozygous (ta2/ta2) or wild-type plants. In the reciprocal crossesbetween a maternal heterozygote and a paternal wild-type, 47.3% (n=188)of F1 grains showed the ta2 mutant phenotype, while in the reciprocalcross between a maternal ta2 plant and a paternal heterozygote, 99.3%(n=425) of F1 individuals showed the ta2 mutant phenotype. From theseresults, it was concluded that the TA2 gene conferred the phenotype by agametophytic, maternal mode of inheritance.

According to the above genetic analyses, a model of ta2 inheritance canbe proposed, according to a gametophytic maternal mode of inheritance.When the genotype of the maternal gametophyte is ta2, the endospermphenotype is the mutant ta2 (thickened aleurone) and is independent ofthe paternal genotype. Therefore, the thick aleurone phenotype wasdetermined solely by the genotype of the maternal gametophyte during thedevelopment of the triploid starchy endosperm and aleurone, such that amaternal heterozygote resulted in 50% of progeny having a thick aleuronephenotype, independent of the paternal genotype, and a maternal ta62/ta2homozygote resulted in 100% of progeny having a thick aleuronephenotype. Further experiments are needed to test whether thegametophytic maternal effect of TA2 is caused by the presence of theadditional copy of the maternal gene in the triploid endosperm or by theeffect of gene imprinting by the maternal gametophyte to suppress theexpression of a paternal TA2 gene.

Example 4. Identification of the Ta2 Gene by Genetic Mapping andSequence Analysis

Identification and Use of SSR and INDEL Markers for Gene Mapping

For gene mapping, an F2 population of plants was produced from thegenetic cross between a plant containing the ta2 mutation in the geneticbackground of ZH11 (a Japonica variety) and a plant of the Indicavariety NJ6. To identify genetic markers which were polymorphic betweenZH11 and NJ6 and which could then be used in the gene mapping, a set ofPCR experiments was performed on leaf DNA samples from homozygous ZH11plants, homozygous NJ6 plants and a 1:1 mixture of the DNAs. Analysis ofthe PCR products by gel electrophoresis allowed comparison of theproducts from ZH11, NJ6 and the mixtures to identify polymorphicmarkers. Primer pairs were selected for the gene mapping only if theamplifications with separate ZH11 and NJ6 DNAs showed discrete anddifferent amplified products and the mixed DNA showed the combination ofboth products. A total of 124 primer pairs were thereby selectedincluding 54 insertion-deletion. polymorphisms (INDEL) and 70 shortsequence repeats (SSR) polymorphisms. These genetic markers weredistributed at approximately 3-4 Mbp intervals along the rice genome andgave good coverage for gene mapping.

For genetic mapping of the ta2 allele, 143 plants from the F2 populationwere scored with the 124 polymorphic markers. Homozygosity of theindividual F2 plants in the mapping population for the aleuronephenotype was assessed carefully by phenotyping of F3 progeny grainsobtained from each F2 plant. Leaf DNA was extracted as described inExample 1. PCR amplifications were done as described in Example 1 andthe products separated by gel electrophoresis through 3% agarose. It wasconcluded from the results that the ta2 locus was located betweenmarkers INDEL 127 and INDEL 129 on Chromosome 1 (FIG. 1, uppermostline), which from the genome sequence of rice corresponding to aphysical distance of approximately 217 kb.

Another 5000 F2 plants were screened with this pair of markers. 362individuals were identified and selected which exhibited a recombinationbetween INDEL 127 and INDEL 129. When these recombinant plants werephenotyped, the ta2 locus was thereby mapped to a 42.8 kb region whichlay between the INDEL 149 and INDEL 128 markers (FIG. 1, second line).

To obtain the nucleotide sequence of this region in the ta2 mutantplants and compare it to the wild-type sequence and thereby identify amutation corresponding to ta2, primers flanking the genomic region weredesigned and DNA sequencing was carried out. The comparison of thegenomic DNA sequences identified two single-nucleotide polymorphisms(SNPs) in the sequenced region, both in the gene annotated asLOC_01g11900. The first was a single nucleotide G (wild-type) to A (ta2)polymorphism at nucleotide position Chr1: 6451738, with reference to therice genome sequence of the Japonica variety, located in intron 14between exon 14 and exon 15 of the gene LOC_01g11900 in chromosome 1(asterisk in FIG. 1). The second polymorphism was a G (wild-type) to A(ta2) substitution at position Chr1: 6452308, which was located in theintronic region (intron 15) between exons 15 and 16 of the geneLOC_01g11900 in chromosome 1.

Upon RNA extraction, reverse transcription and sequencing of the cDNAcorresponding to the ta2 allele, it was observed that the first G to Apolymorphism at Chr1: 6451738 was associated with an insertion of 21 bp(FIG. 2) between exon 14 and exon 15, corresponding to an in-frameinsertion of seven amino acids in the predicted amino acid sequence(FIG. 3). In contrast, there was no change in the cDNA sequence for themutation between exons 15 and 16, corresponding to the secondpolymorphism at position locus Chr1: 6452308. It was concluded fromthese data that the first polymorphism in intron 14 was the causativechange, i.e. the ta2 mutation in that grain. This conclusion wasconfirmed in the Examples below. It was also concluded that the mutationled to a change in the splicing pattern of the RNA transcript of the ta2gene relative to the wild-type Ta2 gene, thereby causing the ta2phenotype. From the ratio of the number of cDNAs having the 21nucleotide insertion to the number of the cDNAs lacking the insertion,it was estimated that about 80% of the RNA transcripts from the ta2(mutant) gene were spliced at the newly created splice site. Presumingthat the mutant polypeptide having the 7 amino acid insertion wasinactive, it was concluded that the mutant ta2 gene retained about 20%of the activity relative to the wild-type.

The gene at position LOC_01g11900 in chromosome 1 of the rice genome hasbeen annotated as the rice ROS1a gene (OsROS1a), a homolog of theArabidopsis thaliana demeter gene (AtDME) which encodes a bifunctionalDNA glycosylase/lyase. The Arabidopsis DME enzyme acts as a DNAdemethylase, reducing methylation of C residues in DNA. Therefore theTa2 gene is synonymous with OsROS1a gene and a homolog of theArabidopsis DME gene.

The nucleotide sequence of the rice ROS1a gene is shown in SEQ ID NO:9,including a promoter and 5-UTR (untranslated region) of 4726nucleotides, a protein coding region from nucleotides 4727-15869including 16 introns, and a 3′UTR of 615 nucleotides. The nucleotidepositions of the 16 introns are provided in the legend to SEQ ID NO:9.SEQ ID NO:9 also includes at its 3′ end a downstream region of 401nucleotides which is not considered to be part of the OsROS1 gene. Thenucleotide sequence of the cDNA corresponding to wild-type OsROS1 geneis provided in SEQ ID NO:8, and the encoded polypeptide of 1952 aminoacids is provided as SEQ ID NO:2.

Rice has four ROS1 genes which encode polypeptides designated OsROS1a,OsROS1b (LOC_Os02g29230), OsROS1c (LOC_Os05g3735) and OsROS1d(LOC_Os05g37410). Rice also has two other Demeter homologs which arethought to encode DNA glycosylases, namely Demeter-like-2 (DML2) andDemeter-like-3 (DML3).

Description of the Structural Features in the Wild-Type Rice TA2Polypeptide

After finding that the rice TA2 gene was the same as OsROS1a, the OsTA2(OsROS1) polypeptide amino acid sequence was examined. Several typicalDNA glycosylase structural features were identified. The glycosylasedomain of ROS1 proteins has at least three identified motifs which aresufficiently conserved to be recognisable: the helix-hairpin-helix (HhH)motif (represented by, for example, amino acids 1491-1515 in OsTA2), aglycine/proline-rich motif followed by a conserved aspartic acid (GPD),and four conserved cysteine residues (for example in the region of aminoacids 1582-1598) to hold a [4Fe-4S] cluster in place. There was also alysine-rich domain (represented by, for example, amino acids 87-139 inOsTA2). Unlike other members of the HhH DNA glycosylase superfamily,ROS1-family members contain two additional conserved domains (domains Aand B) flanking the central glycosylase domain (Mok et al., 2010). Inthe rice TA2 polypeptide (SEQ ID NO:2), domain A occurs at amino acids859 to 965, the glycosylase domain occurs at amino acids 1403 to 1616,and domain B occurs at amino acids 1659 to 1933. Domain A contains arepetitive mixed-charge cluster at amino acids 882-892. It has beenreported that the conserved DNA glycosylase domain of AtDME and theflanking domains A and B are necessary and sufficient for DNAglycosylase/lyase enzymatic activity, as shown by mutagenesis analysis(Mok et al., 2010).

Example 5. Analysis of Nutritional Components in Ta2 Mutant Grain

To measure the composition of mutant grain, particularly fornutritionally important components, ZH11 and ta2 plants were grown atthe same time and under the same conditions in the field. Whole grainflour samples were prepared from grain harvested from the plants andused for the compositional analysis. The results (means of duplicatemeasurements) of the proximate analyses of the flours are given in Table2.

TABLE 2 Compositional analysis of rice grain (in g/100 g of grain) TotalTotal Soluble Insoluble Moisture Ash Protein Total Fat Starch SugarsTotal Fibre Fibre Fibre ZH 11 9.54 1.79 15.3 3.30 66.0 1.02 1.6 0.7 2.9ta2 8.99 2.27 15.6 4.96 60.3 2.58 4.9 0.4 3.9

The proximate analyses indicated an increase of about 50% in the totalfat content in the ta2 mutant flour. Total nitrogen analyses showed nosignificant change in the protein levels between the ta2 mutant andwild-type grains. Ash assays, which measured the amount of materialsleft behind after combustion of dehumidified flour samples, demonstratedan increase of 26% in ta2 grain relative to wild-type. The total fibrelevel increased by about 200% in ta2 grain. The starch content decreasedby 8.6% in ta2 grain relative to wild-type. These data demonstrated thatthe increase in thickness of the aleurone layer in the ta2 mutant causedan increase in the level of aleurone-rich nutrients such as lipid,minerals and fibres without changing the size of the seed. In order tounderstand these and other changes in greater detail, more extensiveanalyses were done as follows.

Minerals

To measure mineral contents, ICP-AES was used which combines inductivelycoupled plasma (ICP) with atomic emission spectrometry (AES) techniques.This is a standard method for measuring mineral content, providing asensitive and high throughput quantitation of a large number of elementsin a single analysis. The data obtained from the analysis showed thatthe mutant grain had levels of zinc and iron which were increased byabout 15% on a weight basis relative to the wild-type grain. Zinc levelsincreased from 13.9 mg/kg to 16.0 mg/kg, while iron increased from 12.4mg/kg to 14.2 mg/kg.

Increases in potassium, magnesium, phosphorus and sulphur were alsoobserved, being increased by about 28%, 23%, 22% and 9%, respectively.These results were consistent with the increase in ash content in ta2grain, which measures mostly minerals.

Antioxidants

Antioxidants are biomolecules capable of counteracting the negativeeffects of oxidation in animal tissues, thus protecting againstoxidative stress-related diseases such as inflammation, cardiovasculardisease, cancer and aging-related disorders (Huang, 2005).

The antioxidant capacity in flours obtained from the ta mutant andwild-type rice grain was measured by an oxygen radical absorbancecapacity (ORAC) assay as described in Example 1. In the ORAC assay, theantioxidant capacity is represented by the competition kinetics betweenendogenous radical scavenging biomolecules and the oxidisable molecularfluorescent probe fluorescein, against the synthetic free radicalsgenerated by AAPH (2,2′-azobis(2-amidino-propane) dihydrochloride). Thecapacity was calculated by comparison of the area under the kineticcurve (AUC), representing the fluorescence degradation kinetics of themolecular probe fluorescein for the grains with the AUC generated byTrolox standards (Prior, 2005). An alternative approach to quantifyingantioxidant capacity is through the use of the Folin-Ciocalteau reagent(FCR); this represents the antioxidant capacity by measuring thereducing capacity of the total phenolic compounds in the food sample.The FCR assay is relatively simple, convenient and reproducible.However, the more time-consuming ORAC assay measures more biologicallyrelevant activity. Since antioxidants include a wide range ofpolyphenols, reducing agents and nucleophiles, measurement by both FCRand ORAC can provide a better coverage and more comprehensiverepresentation of the total antioxidant capacity. As reported by Prior(2005), the results of FCR assay and ORAC measurement are usuallyconsistent.

Both of the FCR and ORAC assays showed increased antioxidant capacity offlour from the ta2 mutant whole grain flour relative to wild-type, ZH11whole grain flour. FCR demonstrated an increase of about 35% in totalphenolic compounds in the ta2 mutant. There was also an 83% increase inhydrophilic antioxidant content in the flour from the ta2 mutant.

Phytate

When grown under conditions with adequate phosphorus, about 70% of totalphosphorus content in rice grain is in the form of phytate or phyticacid (myo-inostitol-1,2,3,4,5,6-hexakisphosphate). Dietary phytate mayalso have beneficial roles for health as a strong antioxidant(Schlemmer, 2009). Total phytate analyses showed an increase in phytatecontent about 19% in ta2 as compared to the wild-type, increasing fromabout 10.8 mg/g to about 12.7 mg/g.

B Vitamins

Levels of the vitamins B3, B6 and B9 in the ta2 flour were higher thanthose in wild-type flour by about 19%, 63% and 58%, respectively. Whenthe assay was repeated with four replicates, the mean increases wereabout 20%, 33% and 38%, respectively. Aleurone was known to be richer invitamins B3, B6 and B9 than endosperm (Calhoun, 1960), so the increasein aleurone thickness in the ta mutant grains was concluded to beresponsible for the increase in vitamin B3, B6 and B9 contents.

Dietary Fibre

Total dietary fibre measured as described in Example 1 as was observedto increase by about 70%. Insoluble fibre increased by about 55%.

Carbohydrates

There was a 9% decrease in the starch content of the ta2 grain on aweight basis. In contrast, sucrose levels increased by 2.5-fold in themutant grain, and monosaccharides (arabinose, xylose, galactose,glucose) were increased from 31% to 118% relative to the wild-type.

CONCLUSIONS

The nutritional analyses showed that wholegrain flour produced fromfield-grown ta2 grain was significantly increased relative to wild-typein most of the aleurone-rich nutrients including the macro-nutrientssuch as lipid and fibre, micronutrients such as minerals (iron, zinc,potassium, magnesium, phosphorus, sulphur), B vitamins such as B3, B6and B9, antioxidants, and aleurone-associated biomolecules such asphenolic compounds and phytate. There was also a substantial increase infree sucrose and monosaccharides. Concomitant with the increase in thesenutrients and micronutrients was a small decrease in starch content inthe ta2 mutant, as a relative percentage.

Example 6. Screening for Additional Mutant Alleles in the Ta2 Gene

The mutagenised population of rice plants in the ZH11 genetic background(Example 2) were screened by TILLING assays as described in Example 1 toidentify further polymorphisms in the Ta2 gene, so that they could betested for a thickened aleurone phenotype. The method usedheteroduplexing of labelled wild-type RNA and candidate mutant RNA withdigestion by endonuclease Cell essentially as described by Jiang et al.(2013). The 5′ region of the TA2 gene was chosen for screening first ofall, but any region of the gene could have been chosen.

Numerous single nucleotide polymorphisms were identified in the 5′region of the Ta2 gene by the TILLING assays. Grains from the plantshaving the polymorphisms were examined for thickened aleurones and othergrain phenotypes as for the first ta2 mutant. Some grains exhibitedthickened aleurones. Those grains that exhibited mutant phenotypes wereselected and progeny plants obtained from them. The nucleotide sequenceof the Ta2 gene in each was determined, confirming the presence of themutations. The altered nucleotide(s) in the Ta2 gene in each mutant wasidentified. Another three thick aleurone mutants are also beingsequenced. Other grains which contained polymorphisms but which did notexhibit thickened aleurones were also identified and maintained forcomparison. The mutants and the other polymorphic lines, and theiraleurone phenotype, that were identified are summarised in Table 3. Themutants having thickened aleurone are shown as: ++, greatly thickenedaleurone; +, weakly thickened aleurone; −, unaltered aleurone phenotype.It was clear that a variety of mutations and resultant phenotypes wasobtained.

The aleurone in wild-type ZH11 grain showed one cell layer in thickness.In contrast in the specific mutants, the aleurones in the mutant grainscomprising the V441A mutation were thickened in the dorsal side,comprising about 5-6 cell layers. The aleurones in mutant S1357F grainswere about 4-5 cell layers in thickness and the grains were shrunken,whereas the aleurones of mutant R482K grains were 2-3 cell layers thickand the grains were not shrunken. The aleurones in mutant S214F grainscontained 2-4 cell layers and the grains were shrunken, as were thegrain from mutants S156F and S1413N. In contrast, the aleurones of K501Sgrains had 2-3 cell layers and its grains were not shrunken. Therefore,a variety of mutants and phenotypes were readily obtained in the Ta2gene.

TABLE 3 Mutations identified in rice Ta2 gene. Mutation Gene Thick Seeddesignation region Mutation aleurone phenotype A1810 Exon S156F ++shrunken B19 Exon S214F ++ shrunken A155 Exon S1413N ++ shrunken A1774Exon A441V ++ normal A2918 Exon S1357F ++ shrunken D11253 Exon K501S +normal A775 Exon R482K + normal A1711 Exon To be determined ++ D11190Exon To be determined ++ B857 Exon To be determined + D11080 Exon D3V −A654 Exon T221I − D113281 Exon P883S − D10394 Exon P843C − A3033 ExonA78V − B1193 Exon E123K − D11321 Exon R487K − A790 Exon R530K − D11029Exon D1425N − A2004 Exon S1272N − A1152 Exon P1225L − A2435 Exon R1390N− B696 Exon synonymous − D11283 Exon synonymous − D11184 Exon synonymous− D11253 Exon synonymous − A1687 Exon synonymous − B1339 Exon synonymous− B1979 Exon synonymous − B2089 Exon synonymous − A3033 Exon A78V −

Of the 60 newly identified lines having polymorphisms in the TA2 gene,19 had amino acid changes (substitutions) in the predicted polypeptideproducts. Of those, at least 7 exhibited thickened aleurone phenotypes.The S1413N and D1425N mutations lay within the glycosylase domain; theother identified mutations lay outside of the glycosylase domain. Apartfrom the initial splice-site variant mutant, all of the identifiedmutations were amino acid substitutions. None were deletions or stopcodons, leading the inventors to conclude that null mutations in OsROS1might be lethal. It has been reported that in Arabidopsis, maternal dinemutations resulted in aborted seeds (Choi et al., 2002 and 2004). Inrice, the presence of a ros1a maternal null allele resulted in earlystage endosperm developmental failure regardless of the paternalgenotype (Ono et al., 2012).

The recovery of ten new, independent mutant alleles in the TA2 (OsROS1a)gene, each of which had a thickened aleurone layer in the grain,indicated conclusively that the mutations in this gene had caused thethick aleurone phenotype. Furthermore these new mutations were all in adifferent region of the gene to the first ta2 mutation indicating thatthe gene could be altered in various positions along the full-lengthgene to achieve the thick aleurone phenotype.

Several of the ta2 genes from the mutants showing thick aleurones arecloned and the encoded polypeptides are expressed and tested for DNAglycosylase/lyase activity. This confirms that the polypeptides havereduced DNA glycosylase/lyase activity compared to the wild-typepolypeptide.

Example 7. Complementation Analysis of the Ta2 Mutant

In order to strengthen the conclusion that mutations in the Ta2(OsROS1a) gene were responsible for the thickened aleurone andassociated phenotypes, complementation experiments were performed byintroducing a wild-type copy of the gene into the mutant line bytransformation. To construct the transformation plasmid for thecomplementation experiment, a 16,882 nucleotide DNA fragment (nucleotidesequence provided as SEQ ID NO:9) including the Ta2 gene was isolatedfrom the wild-type rice genome. This fragment contained, in order, a4726-bp upstream sequence which was considered to contain the promoterof the gene, the entire OsTA2 protein coding region including all of theintrons, a 615 nucleotide 3′-UTR and a 401-bp downstream region. It wasamplified from ZH11 genomic DNA using a series of oligonucleotideprimers, assembled, and then digested with KpnI and SalI and ligated tothe binary vector pCAMBIA1300. That vector also contained a hygromycinresistance gene as a selectable marker gene. The plasmid fortransformation and a control plasmid (empty vector) were each introducedinto Agrobacterium tumefaciens strain EHA105 and used to transform ricerecipient cells using the method as described by Nishimura et al.(2006). A total of 32 T₀ transgenic plants were regenerated from thetransformation with the wild-type Ta2 gene. These plants weretransferred to soil and grown to maturity in a growth chamber. When PCRwas used to test for the presence of the hygromycin resistance gene, 20transformant lines were identified and selected which carried thehygromycin gene. These were grown to maturity and grain (T1 seed)harvested from each plant. Each of these plants contained the T-DNA fromthe vector containing the wild-type Ta2 gene as demonstrated by PCRassays.

Grains harvested from these plants were examined for their aleuronephenotype by staining with Evans blue. At least three of the transformedplants produced grains with normal aleurones like the wild-type,indicating positive expression of the introduced gene and thereforecomplementation of the ta2 mutation. This conclusively proved that themutations in the Ta2 gene caused the mutant phenotypes.

The Ta2 gene is referred to hereinafter as the ROS1a gene; these termsare interchangeable.

Example 8. In Vitro Enzyme Activity Assays of Recombinantly ExpressedTA2 and ta2 Proteins

As described in Example 4 above, the Ta2 gene in rice was the same asthe OsROS1a gene, which is homologous to the Arabidopsis thaliana DNAdemethylase/glycosylase named as Demeter (DME; Gehring et al., 2006).DME breaks the phosphodiester linkage on the 3′ side of a5-methylcytosine residue in a hemi-methylated DNA substrate.

The enzyme activity from recombinantly expressed rice Ta2 and ta2proteins is therefore tested by measuring their activity on ahemi-methylated DNA substrate which has been labelled, to generateend-labelled DNAs that migrate on denaturing polyacrylamide gels at thepredicted position for p elimination products, as described by Gehringet al. (2006).

In order to recombinantly express and purify the Ta2 and ta2polypeptides, full-length ROS1a cDNAs from the wild-type and mutant ta2plants are used as templates in a PCR reaction with oligonucleotidesJH021 (5′-TTAATCTAGAATGCAGAGCATTATGGACTCG-3′; SEQ ID NO:42) and JH017(5′-CGGTCGACTTAGGTTTTGTTGTTCTTCAATTTGC-3′; SEQ ID NO:43), which add XbaIand SalI restriction sites, respectively, to the ends of the amplifiedDNA fragment. The PCR products are digested with XbaI and SalI andcloned into the pMAL-c2× vector (NEB) to create c2×-ROS1a geneticconstructs. The genetic constructs are transformed into E. coli Rosettacells (Novagen). To produce the polypeptides, transformed cells aregrown at 28° C. in LB supplemented with 0.2% glucose, 100 μg/mL ofampicillin and 50 μg/mL of chloramphenicol until an OD600 of 0.4 isreached. ROS1a-Mal fusion protein expression is induced with 10 μM ofIPTG at 18° C. for 1 hr. The cultures are centrifuged at 6,500 rpm for15 min at 4° C. and the pellet is resuspended in 30 mL of 4° C. columnbuffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA). Cells aresonicated for 2 min on ice using a Branson Sonifier 250 at output powersetting of 4. The lysates are centrifuged at 9,000 rpm for 25 min at 4°C. and the supernatants collected and subjected to gravity columnpurification. The ROS1a-Mal fusion proteins are purified through amyloseresin following the manufacturer's protocol (New England Biolabs).Eluted proteins are dialyzed in the Slide-A-Lyzer dialysis cassette(10,000 MWCO; Pierce) against 50% glycerol at 4° C. overnight. Proteinconcentrations are determined by the Bradford method using the ProteinAssay kit (Bio-Rad Laboratories) and the proteins stored at −20° C.until further use.

The ROS1a-Mal fusion proteins are assayed for DNA glycosylase activityagainst hemi-methylated double-stranded DNA substrates as described inExample 1 (Gehring et al., 2006).

As controls, no lyase activity or covalent trapping is detected whenROS1a is incubated with non-methylated DNA oligonucleotides or whenhemi-methylated DNA substrate is incubated in the absence of enzyme.

Example 9. Homologues of the ROS1a Gene in Rice

The plant genes that encode DNA glycosylases which mediate DNAdemethylation have been characterised mainly in Arabidopsis thaliana(Chan et al., 2005; Law and Jacobsen, 2010; Zhu, 2009). They include theDemeter (DME, Choi et al., 2002; Gehring et al., 2006), ROS1 (Gong etal., 2002; Agius et al., 2006), Demeter-like 2 (DML2) and Demeler-like 3genes (DML3, Choi et al., 2002; Ortega-Galisteo et al., 2008). Thelargest of these genes (and encoded polypeptides), DME, is expressedmost strongly in the homodiploid central cell of the female gametophytebefore fertilisation where it promotes maternal allele-specific globalhypomethylation and expression of imprinted genes including in theendosperm. In contrast, ROS1, DML2 and DML3 are expressed in vegetativetissues (Gong et al., 2002; Penterman et al., 2007). Compared with ROS1,the level of expression of the DML2 and DML3 genes was low (Mathieu etal., 2007). Moreover, homozygous mutations in ros1, dml2 and dml3yielded no obvious morphological phenotypes whereas a maternal dmemutation resulted in aborted seeds, i.e. embryo lethal, and was nottransmitted to progeny (Choi et al., 2002 and 2004). Despite their lowexpression levels, the ROS1, DML2 and DML3 polypeptides still functionas DNA glycosylases/lyases (Gong et al., 2002; Morales-Ruiz et al.,2006; Penterman et al., 2007). From this data, one would not haveexpected a ROS1 mutation to have caused a thickened aleurone phenotype.

Phylogenetic analysis revealed that the rice genome encodes 6 putativeDNA glycosylases for cytosine demethylation, including four that appearto be ROS1 orthologs (OsROS1a, OsROS1b, OsROS1c, OsROS1d) and twoapparent DML3 orthologs (Zemach et al., 2010). A null mutation inOsROS1a was identified but was not transmitted from either male orfemale plants containing the mutation to progeny, presumably becauseROS1a wild-type DNA glycosylase is indispensible in both male and femalegametophytic development (Ono et al., 2012). The inventors are not awareof any published reports of partial mutations in OsROS1a.

The three identified motifs in the DNA glycosylase domain, namely thehelix-hairpin-helix (HhH) motif, a glycine/proline-rich motif followedby a conserved aspartic acid (GPD), and the four conserved cysteineresidues (Example 4) were present in each member of the Demeter family.The glycosylase domain structure was also found in human 8-oxoguanineDNA glycosylase (hOGG1), E. coli adenine DNA glycosylase (MutY), andendonuclease III (Endo III) (Bruner et al. 2000; Guan et al. 1998; Moket al., 2010). Unlike other members of the HhH DNA glycosylasesuperfamily, DME-family members contained two additional conserveddomains (domain A and domain B) flanking the central glycosylase domain(Mok et al., 2010).

The nucleotide sequences of the protein coding regions for thehomologous genes were aligned by ClustalW(www.ebi.ac.uk/Tools/msa/clustalw2/). The extent of sequence identity ofthe rice ROS1a protein coding region to the corresponding region of thehomologous genes in other species is shown in Table 4.

The inventors concluded from these analyses that rice has multiple ROS1gene homologs but no DME genes. In rice, as for Arabidopsis, ROS1 wasclearly distinguishable from its homologs DML2 and DML3 in the samespecies in terms of the extent of sequence identity.

TABLE 4 Nucleotide sequence identity to coding region of rice OsROS1a orTaROS1a-5B. Identity to Gene Accession Number OsROS1a OsROS1aLOC_Os01g11900  100% OsROS1b LOC_Os02g29230 41.7% OsROS1c LOC_Os05g3735042.0% OsROS1d LOC_Os05g37410 41.9% OsDML3a LOC_Os02g29380 34.9% OsDML3bLOC_Os04g28860 33.2% AtDME NM001085058.1 40.6% AtROS1 NM129207.4 41.5%AtDML2 NM111836.5 39.2% AtDML3 NM119567.3 40.4%

Example 10. Expression of ROS1a Gene in Rice

Experiments were carried out to analyse expression of the TA2 gene indifferent rice tissues, including in parts of the developing grain. In afirst experiment, TA2 mRNA was detected in rice tissue sections by insitu hybridisation as described by Brewer et al. (2006). Briefly,various rice tissues were fixed in FAA fixative for 8 h at 4° C. aftervacuum infiltration, dehydrated using a graded ethanol series followedby a xylene series, and embedded in Paraplast Plus (Sigma-Aldrich).Microtome sections (8 μm) were mounted on Probe-On Plus microscopeslides (Fisher).

From the hybridisation signals, it was concluded that TA2 was expressedin the pericarp, testa and aleurone tissues and in the starchy endospermof rice, but not in the vascular bundle.

Realtime reverse transcription polymerase chain reaction (RT-PCR) wasused to assay relative expression levels in different plant tissues.Surprisingly, the results indicated highest relative expression inpollen, followed by anthers, young panicles and aleurone tissue (FIG.6). It was considered that the specific expression of OsROS1a in anthersmight be involved in the suppression of transposons in the malegametophyte. In Arabidopsis tricellular pollen, active DNA demethylationis important in maintaining a basal expression of transposons invegetative cell nuclei so as to produce siRNA for reinforcing RNAdependent DNA methylation (RdDM) of the transposons in male gametes,i.e. the two sperm cells (Zhu, 2009; Zhu et al., 2007).

Expression of ROS1a in the developing seed increased to 10 days postanthesis and then declined thereafter. Strong expression was observed inboth the starchy endosperm and aleurone tissues. The expression patternearly in seed development was consistent with the formation of thickenedaleurone, prior to cellularisation of the endosperm during seeddevelopment. The inventors concluded that reduced expression of ROS1a inthe period from the day of anthesis to 7 days post anthesis(pollination) (0-7 DAP) was critical to formation of the thick alerone.

Example 11. Patterns of Gene Methylation in Rice

To determine the patterns of methylation of all rice genes,collectively, in the ta2 mutant plants relative to the wild-type TA2plants, DNA was isolated from endosperms and embryos and treated withbisulfite which reacts with unmethylated cytosines, followed by Illuminasequencing. Endosperms were isolated at 10 DAP from the developing ricegrains of the ta2 and wild-type (ZH11) plants, and embryos from thewild-type plants at the same stage of grain development. For sequencingfollowing bisulfite treatment, custom Illumina adapters were synthesizedin which cytosines were replaced by 5-methylcytosines, so that theadapters would survive the bisulfite conversion. Paired end (PE)adapters were synthesized which allowed each molecule to be sequencedfrom both ends, thus facilitating subsequent alignment to the genomicscaffold sequence. About 0.5-1 μg of genomic DNA was isolated fromendosperms dissected from each of the wild-type and ta2 plants as wellas from wild-type embryos. The isolated DNA preparations were sheared bysonication to fragments of 100-500 bp. The adapters were ligated to thesheared fragments following the Illumina protocol. The DNAs were thentreated twice with sodium bisulfite, which converts unmethylatedcytosines (C) to uridines (U), using the Qiagen EpiTect kit andamplified by 18 cycles of PCR using PfuTurboCx DNA polymerase(Stratagene), a proofreading enzyme that tolerates uridines in thetemplate strand. This PCR amplification resulted in a library of DNAfragments with distinct adapters at each end, so that the ‘forward’Illumina sequencing primer yielded a nucleotide sequence from the‘original’ genomic DNA-derived strand (where a C corresponded to amethylated C, and a T corresponded to a non-methylated C where a Coccurred in the genomic sequence), and the ‘reverse’ Illumina sequencingprimer produced a nucleotide sequence from the complementary strand(where a G corresponded to a methylated C on the opposite strand, and anA corresponded to a non-methylated C where a C occurred in the genomicsequence).

The extent of CG and CHG methylation in the DNA obtained from the ta2endosperms was greater than that in the DNA obtained from the controlZH11 endosperms, indicating that the mutation of TA2 (OsROS1a) reducedthe demethylation process in rice endosperm, whereas the extent of CHHmethylation in the ta2 endosperm was not significantly different to thatin wild-type ZH11 endosperm.

Example 12. Further Analysis of Nutritional Components in Ta2 MutantGrain

Further analyses were carried out to measure the nutritional componentsof mutant grain compared to the corresponding wild-type grain (ZH11),grown at the same time and under the same conditions in the field. Wholegrain flour samples were prepared from the grain harvested from theplants and used for compositional analysis as described in Example 5.The results of the proximate analyses of the flours for grain grown inAustralia are given in Table 5. The results for grain grown in China aregiven in Table 6.

The proximate analyses indicated an increase of about 50% in the totallipid content in the ta2 mutant flour. Total nitrogen analyses showed asignificant change in the protein levels between the ta2 mutant andwild-type grains in China but not in Australia, which may have due todifferent nitrogen fertiliser regimes. The total fibre level increasedby about 66% or 91% in ta2 grain. The starch content decreased by 9% inta2 grain relative to wild-type. These data confirmed that the increasein thickness of the aleurone layer in the ta2 mutant caused ansignificant increase in the levels of aleurone-rich nutrients such aslipid, minerals and fibres without changing the size of the seed. Eventhough the absolute numbers differed in the two growth environments, therelative increases in ta2 grain were reasonably consistent.

TABLE 5 Composition of ros1a mutant rice grain (Australia) compared towild-type % Component Units ZH11 ta2 change Total Starch g/100 g 67.961.8 −9% Fibre Total Dietary fibre g/100 g 3.45 5.73 66% Soluble DietaryFibre g/100 g 0.54 0.56  4% Insoluble Dietary Fibre g/100 g 2.74 4.2655% B Vitamins Niacin (Vitamin B3) mg/100 g 6.53 7.90 21% Pyridoxine(Vitamin B6) mg/100 g 0.10 0.13 33% Folate (Vitamin B9) μg/100 g 19.425.6 32% Mineral Total Ash g/100 g 1.79 2.39 33% Iron mg/kg 12.4 14.214% Zinc mg/kg 13.7 16.0 17% Potassium mg/kg 3,930 4,780 22% Magnesiummg/kg 1,270 1,560 23% Sulphur mg/kg 1,240 1,350  9% Simple sugar Sucroseg/100 g 0.95 2.54 169%  NNSP Total mg/100 mg 1.54 2.48 61% NNSPArabinose mg/100 mg 0.28 0.62 61% components Xylose mg/100 mg 0.26 0.5089% Mannose mg/100 mg 0.11 0.16 59% Galactose mg/100 mg 0.10 0.20 53%Glucose mg/100 mg 0.77 1.01 47% Protein g/100 g 15.18 15.26  1% Phytatemg/g 10.79 12.69 18% Phenolics Total Phenolics μg/g 3,180 4,570 43% FreePhenolics μg/g 529 665 26% Conjugated Phenolics μg/g 348 692 99% BoundPhenolics μg/g 2,250 2,950 31% Antioxidants ORAC μmol/g 12.3 22.6 84%Moisture 9.5 8.9 −6% Lipid Total lipid g/100 g 3.29 4.95 50% Lipid Fattyacid 18:0 5.1% 4.5% −13%  composition Fatty acid 18:1n9t 3.3% 2.6% −22% Fatty acid 18:1n9c 32.7% 43.2% 32% Fatty acid 18:1n7 1.7% 1.4% −19% Fatty acid 18:2n6 36.0% 27.9% −22% 

TABLE 6 Composition of ros1a mutant rice grain (China) compared towild-type Specific Component component ZH11 ta2 % change Protein totalprotein 12.38 14.12 14.05 Amino acids Asparagine 1.16 1.53 31.90Threonine 0.45 0.54 20.00 Serine 0.64 0.73 14.06 Glutamine 2.29 2.404.80 Glycine 0.59 0.76 28.81 Alanine 0.71 0.84 18.31 Cysteine 0.26 0.3015.38 Valine 0.72 0.79 9.72 Methionine 0.22 0.22 0.00 Isoleucine 0.500.53 6.00 Leucine 1.02 1.05 2.94 Tyrosine 0.59 0.58 −1.69 Phenylalanine0.66 0.69 4.55 Histidine 0.43 0.54 25.58 Lysine 0.43 0.60 39.53 Arginine1.14 1.39 21.93 Proline 0.52 0.59 13.46 Starch amylose 9.80 5.14 −47.55Minerals Selenium (Se) 0.03 0.03 8.78 Calcium 167.89 231.39 37.82 Fe15.24 17.76 16.54 Zn 28.68 41.37 44.25 Antioxidants total 0.06 0.0833.33 flavonoids Fibre Total dietary 3.26 6.23 91.10 fibre VitaminsVitamin A 1.53 5.52 260.78 Vitamin E 0.47 1.00 112.77 Vitamin B1 0.500.57 12.97 Vitamin B2 0.04 0.08 116.67

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

This application claims priority from AU 2015904754 filed 18 Nov. 2015,the entire contents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

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The invention claimed is:
 1. A rice grain comprising an aleurone, astarchy endosperm and a mutant ROS1a gene which encodes a ROS1apolypeptide, wherein the grain is homozygous for the mutant ROS1a gene,and wherein the ROS1a polypeptide comprises an amino acid sequence whichis identical to SEQ ID NO: 1, or comprises an amino acid substitutionwhich is S156F, S214F, S1413N, A441V, S1357F, K501S or R482K withreference to SEQ ID NO:
 2. 2. The grain of claim 1, wherein the graincomprising the mutant ROS1a gene has a reduced total amount of ROS1apolypeptide compared to a wild-type rice grain lacking the mutant ROS1agene in one or more of aleurone, testa and starchy endosperm of thegrain.
 3. The grain of claim 1, wherein the mutant ROS1a gene encodes aROS1a polypeptide which comprises an amino acid sequence which isidentical to SEQ ID NO:
 1. 4. The grain of claim 1, which has beencooked, cracked, par-boiled or heat-stabilised.
 5. The grain of claim 1,wherein the rice grain comprises a ROS1a gene which when expressedproduces a reduced level of a wild-type ROS1a polypeptide relative to awild-type ROS1a gene whose cDNA sequence is provided as SEQ ID NO:
 8. 6.The grain of claim 1, wherein the aleurone of the grain is thickenedcompared to the aleurone from the wild-type rice grain.
 7. The grain ofclaim 6, wherein the aleurone of the grain comprises a number of layersof cells, wherein the number is 2, 3, 4, 5, 6, 7 or
 8. 8. The grain ofclaim 1, wherein the ROS1a polypeptide comprises an amino acidsubstitution which is S156F, S214F, S1413N, A441V, S1357F, K501S orR482K with reference to SEQ ID NO:
 2. 9. The grain of claim 1,comprising a higher mineral content on a weight basis compared to awild-type rice grain lacking the mutant ROS1a gene, wherein the mineralcontent is the content of one or more or all of zinc, iron, potassium,magnesium, phosphorus and sulphur.